- Email: [email protected]
Beneficial Effects of Nerve Growth Factor on Islet Transplantation
Beneficial Effects of Nerve Growth Factor on Islet Transplantation G. Miao, J. Mace, M. Kirby, A. Hopper, R. Peverini, R. Chinnock, J. Shapiro, and E. Hathout ABSTRACT Objective. Development of the Edmonton protocol was a pivotal contribution to clinical islet transplantation (ITx). Persistent limitations to ITx include insufficient supply and posttransplant functional failure of islets. In this study, nerve growth factor (NGF) was used to enhance both cultured and transplanted beta-cell function, thus achieving prolonged graft survival. Methods. Fluorescence microscopy with ethidium bromide and SYTO green staining was used to evaluate balb/c mouse islet viability. Islets were syngeneically transplanted under the kidney capsule of recipients with streptozotocin-induced diabetes. Intraperitoneal glucose tolerance was used to test posttransplant function. Results. Improved viability was found in murine islets cultured for 48 hours in 500 ng/mL NGF (P ⬍ .05). A submarginal islet mass (260 islet equivalents/recipient) was used for ITx. The NGF-culture resulted in prolonged islet survival (24.7 days vs 5.5 days without NFG culture, n ⫽ 6). Intravenous injection of NGF (6 g) on the day of transplant and postoperative days (POD) 1 ⫹ 2 prolonged islet survival from 4.1 days (no treatment) to 13.2 days (n ⫽ 6). Glucose tolerance testing performed at posttransplant day 4 showed improvement at 60 and 120 minutes in recipients treated intravenously with NGF (blood glucose of 95 ⫾ 15 vs 210 ⫾ 78 and 57 ⫾ 6 vs 176 ⫾ 70 mg/dL, respectively). Conclusion. NGF may improve beta-cell function and result in prolonged survival of both cultured and transplanted islets.
T
YPE 1 diabetes is a significant health problem in both childhood and adulthood. Tight glycemic control at the risk of hypoglycemia has been shown to prevent or delay complications.1 Currently, the only way to restore normoglycemia without the associated risk of hypoglycemia is by transplantation of whole pancreas or islets. Less invasive and more targeted surgical and immunosuppressive regimens make islet transplantation (ITx) a more attractive treatment for type 1 diabetes.2 Enhancing beta-cell function can minimize problems of insufficient pancreatic donors and posttransplant failure of islets.3 In vitro, nerve growth factor (NGF)—a neurotrophic factor that stimulates survival, differentiation, and regeneration of neurons by binding to cell-surface receptors—was shown to increase glucosestimulated insulin secretion by activating calcium-channels.4 In the present study, we demonstrate prolonged islet survival in vivo following transplantation of a submarginal islet mass co-cultured with NGF in an experimental model of type 1 diabetes.
MATERIALS AND METHODS Experimental Animals Male balb/c mice, age 8 to 10 weeks (Harlan, Indianapolis, IN), were housed under specific pathogen-free conditions with a 12-hour light/dark cycle and had free access to food and water. The subsequent experimental protocol was approved by the Institutional Animal Care Use Committee.
Islet Transplantation Balb/c recipients were rendered diabetic by intraperitoneal injection of streptozotocin (Sigma-Aldrich, St. Louis, Mo) at a dose of From the Department of Pediatrics (G.M., J.M., M.K., A.H., R.P., R.C., E.H.), Loma Linda University, Loma Linda, California, and Department of Surgery (J.S.), University of Alberta, Alberta, Edmonton, Canada. Address reprint requests to Eba Hathout, MD, Islet Transplant Lab, Department of Pediatrics, Loma Linda University, 11175 Campus St., Coleman Pavilion, Room A1120R, Loma Linda, CA 92354. E-mail: [email protected]
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.057
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3490
Transplantation Proceedings, 37, 3490 –3492 (2005)
NERVE GROWTH FACTOR ON ISLET TRANSPLANT
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Fig 1. (A) Assessment of islet cell viability using fluorescence microscopy with ethidium bromide (dead cells, red) and SYTO green (living cells, green) staining. Islets cultured without NGF show the presence of numerous dead cells (red) at the center of the islets (arrow). Islets cultured with NGF show fewer dead cells. Original magnification, ⫻100. (B) Quantitative evaluation of islet cell viability. 250 mg/kg. Diabetes was confirmed by a capillary blood glucose level greater than 300 mg/dL on 2 separate days. Pancreatic tissue was digested with collagenase type V (Sigma-Aldrich), followed by Ficoll (Ficoll 400, Sigma-Aldrich) density-gradient centrifugation. A total of 260 islet equivalents (IEQ) were handpicked and syngeneically implanted under the kidney capsules of diabetic recipients. Random blood glucose levels exceeding 250 mg/dL on 2 consecutive days were considered evidence of graft failure.
Experimental Protocols Group A: Diabetic recipients transplanted with freshly isolated untreated islets (n ⫽ 7); group B: diabetic recipients, each intravenously injected with 6 g of mouse NGF 2.5S (BD, Bedford, MA) on the day of transplant and the subsequent 2 days (n ⫽ 6); group C: diabetic recipients of islets cultured for 24 hours without NGF before ITx (n ⫽ 6); group D: diabetic recipients transplanted with islets cultured with NGF (500 ng/mL) for 24 hours before ITx (n ⫽ 6).
Islet Viability Fluorescence microscopy was used to evaluate viability of islets cultured in serum-free medium with and without 500 ng/mL of NGF for 48 hours. Aliquots of 50 to 100 islets were stained with ethidium bromide and SYTO green, and the number of islets showing more than 50% viable cells were counted and expressed as percent of total.
Glucose Tolerance Test Glucose tolerance was tested in recipient mice 4 days after transplantation. Following an overnight fast, mice were injected intraperitoneally with D-glucose (2.0 g/kg body weight). Blood samples were taken at various time points (0 –120 minutes).
Statistics Statistical evaluation was performed using the Student t test. Differences among experimental groups were considered significant if P value was ⬍.05. Data were expressed as mean ⫾ standard error (SE).
RESULTS NGF Improves Islet Viability
The viability of islets cultured without NGF was reduced as shown by the high content of dead cells (Fig 1A, control). The number of dead cells was reduced when islets were cultured with NGF (Fig 1A, NGF). NGF improved the percentage of viable islets from 89% to 94% (P ⬍ .05, Fig 1B). Treatment of Islets With NGF Enhances Islet Function Following Transplantation
To test the function of NGF-treated islets in vivo, NGFprecultured islets were transplanted beneath the kidney capsule of diabetic recipients (group D). Alternatively, Table 1. Transplanted Islet Survival in Streptozotocin-Induced Diabetic Recipients Group
Treatment
Survival Days/Recipient
Mean Survival Time (days)
A B C D
None NGF IV Culture without NGF Culture with NGF
2⫻5,3,16 2,6,7,10,12,⬎28 1,2⫻2,3,12,13 2,6,⬎24,⬎35⫻2,⬎46
4.1 13.2 5.5 24.7
Graft failure is defined as blood glucose ⬎250 mg/dL.
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Fig 2. Glucose tolerance test 4 days after islet implantation. Blood glucose levels were monitored before and after glucose injection.
NGF was administered intravenously to diabetic recipients of islet transplants on the day of ITx and postoperative days (POD) 1⫹2 (group B). Culture of islets in NGF prolonged graft survival (24.7 days vs 5.5 days in controls). Comparing group B with group A, 3-day intravenous injection of NGF resulted in prolonged survival (from 4.1 days to 13.3 days, Table 1). Glucose Tolerance
Intraperitoneal glucose tolerance test performed on POD 4 showed amelioration of hyperglycemia in group B in comparison to group A (Fig 2). The effect was most pronounced at 60 and 120 minutes. DISCUSSION
Denervation of transplanted islets may affect their capacity for appropriate metabolic control.5 Both Trk-A and p75NGFR, the high and the low-affinity NGF receptors, are expressed in different insulin-producing (beta) cell lines.6,7 The importance of signals through the Trk-A receptor in the developing nervous system has been confirmed by Trk-A knockouts in transgenic animals.8 These mechanistic studies may be applicable in our transplant model. In the present study, supplementation of culture medium with NGF was demonstrated to improve islet viability. Moreover, using a submarginal islet mass transplant model prolonged islet survival
MIAO, MACE, KIRBY ET AL
following NGF-culture or systemic NGF-treatment of islet recipients. These phenomena may be related to improvement of islet function by reinnervation of transplanted islets beneath the kidney capsule. Superior glucose tolerance results at 60 and 120 minutes might be related to a relatively larger effect of NGF on second rather than first-phase insulin release by transplanted islets. During the isolation process, islets are subjected to severe adverse conditions that potentially impair survival and ultimately contribute to graft failure. Both the integrity of NGF/NGF receptor systems in beta-cells and NGF bioavailability in culture have been shown to play a role in beta-cell survival in culture prior to transplantation.9 In this study, pretransplant culture with NGF optimized posttransplant islet survival, possibly by NGF-mediated inhibition of islet apoptosis. Taken together, the above results indicate that NGF may improve islet function and result in prolonged survival of both cultured and transplanted islets. Such a neurotrophic approach may prove beneficial in human islet transplantation. REFERENCES 1. Chase HP, Lockspeiser T, Peery B, et al: The impact of the diabetes control and complications trial and humalog insulin on glycohemoglobin levels and severe hypoglycemia in type 1 diabetes. Diabetes Care 24:430, 2001 2. Sucherland DE: Current status of beta-cell replacement therapy (pancreas and islet transplantation) for treatment of diabetes mellitus. Transplant Proc 35:1625, 2003 3. Garcia-Ocana A, Vasavada RC, Takane KK, et al: Using beta-cell growth factors to enhance human pancreatic islet transplantation. J Clin Endocrinol Metab 86:984, 2001 4. Rosenbaum T, Sanchez-Soto MC, Hiriart M: Nerve growth factor increases insulin secretion and barium current in pancreatic beta-cells. Diabetes 50:1755, 2001 5. Partis AJ, Rajotte RV, Krukoff TL: Reinnervation of isolated islets of Langerhans transplanted beneath the kidney capsule in the rat. Cell Transplant 3:163, 1994 6. Scharfmann R, Tazi A, Polak M, et al: Expression of functional nerve growth factor receptors in pancreatic beta-cell lines and fetal rat islets in primary culture. Diabetes 43:1829, 1993 7. Polak M, Scharfmann R, Seilheimer B, et al: Nerve growth factor induces neuro-like differentiation of an insulin-secreting beta cell line. Proc Natl Acad Sci U S A 90:5781, 1993 8. Smeyne RJ, Klein R, Schnapp A, et al: Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 368:246, 1994 9. Pierucci D, Cicconi S, Bonini P, et al: NGF-withdrawal induces apoptosis in pancreatic beta cells in vitro. Diabetologia 44:1281, 2001
T
YPE 1 diabetes is a significant health problem in both childhood and adulthood. Tight glycemic control at the risk of hypoglycemia has been shown to prevent or delay complications.1 Currently, the only way to restore normoglycemia without the associated risk of hypoglycemia is by transplantation of whole pancreas or islets. Less invasive and more targeted surgical and immunosuppressive regimens make islet transplantation (ITx) a more attractive treatment for type 1 diabetes.2 Enhancing beta-cell function can minimize problems of insufficient pancreatic donors and posttransplant failure of islets.3 In vitro, nerve growth factor (NGF)—a neurotrophic factor that stimulates survival, differentiation, and regeneration of neurons by binding to cell-surface receptors—was shown to increase glucosestimulated insulin secretion by activating calcium-channels.4 In the present study, we demonstrate prolonged islet survival in vivo following transplantation of a submarginal islet mass co-cultured with NGF in an experimental model of type 1 diabetes.
MATERIALS AND METHODS Experimental Animals Male balb/c mice, age 8 to 10 weeks (Harlan, Indianapolis, IN), were housed under specific pathogen-free conditions with a 12-hour light/dark cycle and had free access to food and water. The subsequent experimental protocol was approved by the Institutional Animal Care Use Committee.
Islet Transplantation Balb/c recipients were rendered diabetic by intraperitoneal injection of streptozotocin (Sigma-Aldrich, St. Louis, Mo) at a dose of From the Department of Pediatrics (G.M., J.M., M.K., A.H., R.P., R.C., E.H.), Loma Linda University, Loma Linda, California, and Department of Surgery (J.S.), University of Alberta, Alberta, Edmonton, Canada. Address reprint requests to Eba Hathout, MD, Islet Transplant Lab, Department of Pediatrics, Loma Linda University, 11175 Campus St., Coleman Pavilion, Room A1120R, Loma Linda, CA 92354. E-mail: [email protected]
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.057
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3490
Transplantation Proceedings, 37, 3490 –3492 (2005)
NERVE GROWTH FACTOR ON ISLET TRANSPLANT
3491
Fig 1. (A) Assessment of islet cell viability using fluorescence microscopy with ethidium bromide (dead cells, red) and SYTO green (living cells, green) staining. Islets cultured without NGF show the presence of numerous dead cells (red) at the center of the islets (arrow). Islets cultured with NGF show fewer dead cells. Original magnification, ⫻100. (B) Quantitative evaluation of islet cell viability. 250 mg/kg. Diabetes was confirmed by a capillary blood glucose level greater than 300 mg/dL on 2 separate days. Pancreatic tissue was digested with collagenase type V (Sigma-Aldrich), followed by Ficoll (Ficoll 400, Sigma-Aldrich) density-gradient centrifugation. A total of 260 islet equivalents (IEQ) were handpicked and syngeneically implanted under the kidney capsules of diabetic recipients. Random blood glucose levels exceeding 250 mg/dL on 2 consecutive days were considered evidence of graft failure.
Experimental Protocols Group A: Diabetic recipients transplanted with freshly isolated untreated islets (n ⫽ 7); group B: diabetic recipients, each intravenously injected with 6 g of mouse NGF 2.5S (BD, Bedford, MA) on the day of transplant and the subsequent 2 days (n ⫽ 6); group C: diabetic recipients of islets cultured for 24 hours without NGF before ITx (n ⫽ 6); group D: diabetic recipients transplanted with islets cultured with NGF (500 ng/mL) for 24 hours before ITx (n ⫽ 6).
Islet Viability Fluorescence microscopy was used to evaluate viability of islets cultured in serum-free medium with and without 500 ng/mL of NGF for 48 hours. Aliquots of 50 to 100 islets were stained with ethidium bromide and SYTO green, and the number of islets showing more than 50% viable cells were counted and expressed as percent of total.
Glucose Tolerance Test Glucose tolerance was tested in recipient mice 4 days after transplantation. Following an overnight fast, mice were injected intraperitoneally with D-glucose (2.0 g/kg body weight). Blood samples were taken at various time points (0 –120 minutes).
Statistics Statistical evaluation was performed using the Student t test. Differences among experimental groups were considered significant if P value was ⬍.05. Data were expressed as mean ⫾ standard error (SE).
RESULTS NGF Improves Islet Viability
The viability of islets cultured without NGF was reduced as shown by the high content of dead cells (Fig 1A, control). The number of dead cells was reduced when islets were cultured with NGF (Fig 1A, NGF). NGF improved the percentage of viable islets from 89% to 94% (P ⬍ .05, Fig 1B). Treatment of Islets With NGF Enhances Islet Function Following Transplantation
To test the function of NGF-treated islets in vivo, NGFprecultured islets were transplanted beneath the kidney capsule of diabetic recipients (group D). Alternatively, Table 1. Transplanted Islet Survival in Streptozotocin-Induced Diabetic Recipients Group
Treatment
Survival Days/Recipient
Mean Survival Time (days)
A B C D
None NGF IV Culture without NGF Culture with NGF
2⫻5,3,16 2,6,7,10,12,⬎28 1,2⫻2,3,12,13 2,6,⬎24,⬎35⫻2,⬎46
4.1 13.2 5.5 24.7
Graft failure is defined as blood glucose ⬎250 mg/dL.
3492
Fig 2. Glucose tolerance test 4 days after islet implantation. Blood glucose levels were monitored before and after glucose injection.
NGF was administered intravenously to diabetic recipients of islet transplants on the day of ITx and postoperative days (POD) 1⫹2 (group B). Culture of islets in NGF prolonged graft survival (24.7 days vs 5.5 days in controls). Comparing group B with group A, 3-day intravenous injection of NGF resulted in prolonged survival (from 4.1 days to 13.3 days, Table 1). Glucose Tolerance
Intraperitoneal glucose tolerance test performed on POD 4 showed amelioration of hyperglycemia in group B in comparison to group A (Fig 2). The effect was most pronounced at 60 and 120 minutes. DISCUSSION
Denervation of transplanted islets may affect their capacity for appropriate metabolic control.5 Both Trk-A and p75NGFR, the high and the low-affinity NGF receptors, are expressed in different insulin-producing (beta) cell lines.6,7 The importance of signals through the Trk-A receptor in the developing nervous system has been confirmed by Trk-A knockouts in transgenic animals.8 These mechanistic studies may be applicable in our transplant model. In the present study, supplementation of culture medium with NGF was demonstrated to improve islet viability. Moreover, using a submarginal islet mass transplant model prolonged islet survival
MIAO, MACE, KIRBY ET AL
following NGF-culture or systemic NGF-treatment of islet recipients. These phenomena may be related to improvement of islet function by reinnervation of transplanted islets beneath the kidney capsule. Superior glucose tolerance results at 60 and 120 minutes might be related to a relatively larger effect of NGF on second rather than first-phase insulin release by transplanted islets. During the isolation process, islets are subjected to severe adverse conditions that potentially impair survival and ultimately contribute to graft failure. Both the integrity of NGF/NGF receptor systems in beta-cells and NGF bioavailability in culture have been shown to play a role in beta-cell survival in culture prior to transplantation.9 In this study, pretransplant culture with NGF optimized posttransplant islet survival, possibly by NGF-mediated inhibition of islet apoptosis. Taken together, the above results indicate that NGF may improve islet function and result in prolonged survival of both cultured and transplanted islets. Such a neurotrophic approach may prove beneficial in human islet transplantation. REFERENCES 1. Chase HP, Lockspeiser T, Peery B, et al: The impact of the diabetes control and complications trial and humalog insulin on glycohemoglobin levels and severe hypoglycemia in type 1 diabetes. Diabetes Care 24:430, 2001 2. Sucherland DE: Current status of beta-cell replacement therapy (pancreas and islet transplantation) for treatment of diabetes mellitus. Transplant Proc 35:1625, 2003 3. Garcia-Ocana A, Vasavada RC, Takane KK, et al: Using beta-cell growth factors to enhance human pancreatic islet transplantation. J Clin Endocrinol Metab 86:984, 2001 4. Rosenbaum T, Sanchez-Soto MC, Hiriart M: Nerve growth factor increases insulin secretion and barium current in pancreatic beta-cells. Diabetes 50:1755, 2001 5. Partis AJ, Rajotte RV, Krukoff TL: Reinnervation of isolated islets of Langerhans transplanted beneath the kidney capsule in the rat. Cell Transplant 3:163, 1994 6. Scharfmann R, Tazi A, Polak M, et al: Expression of functional nerve growth factor receptors in pancreatic beta-cell lines and fetal rat islets in primary culture. Diabetes 43:1829, 1993 7. Polak M, Scharfmann R, Seilheimer B, et al: Nerve growth factor induces neuro-like differentiation of an insulin-secreting beta cell line. Proc Natl Acad Sci U S A 90:5781, 1993 8. Smeyne RJ, Klein R, Schnapp A, et al: Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 368:246, 1994 9. Pierucci D, Cicconi S, Bonini P, et al: NGF-withdrawal induces apoptosis in pancreatic beta cells in vitro. Diabetologia 44:1281, 2001