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. 2012 Jan 1;4(1):24–31. doi: 10.4161/isl.18467

Nerve growth factor is associated with islet graft failure following intraportal transplantation

Yukihiko Saito 1,2, Nathaniel K Chan 1, Naoaki Sakata 3, Eba Hathout 1,*
PMCID: PMC3365801  PMID: 22192949

Abstract

Nerve growth factor (NGF) has recently been recognized as an angiogenic factor with an important regulatory role in pancreatic β-cell function. We previously showed that treatment of pancreatic islets with NGF improved their quality and viability. Revascularization and survival of islets transplanted under the kidney capsule were improved by NGF. However, the usefulness of NGF in intraportal islet transplantation was not previously tested. To resolve this problem, we transplanted syngeneic islets (360 islet equivalents per recipient) cultured with or without NGF into the portal vein of streptozotocin-induced diabetic BALB/c mice. Analysis revealed that 44.4% (4/9) of control and 12.5% (1/8) of NGF-treated mice attained normoglycemia (≤ 200 mg/dL) (p = 0.195). NGF-treated islets led to worse graft function (area under the curve of intraperitoneal glucose tolerance tests (IPGTT) on post-operative day (POD) 30, control; 35,800 ± 3,960 min*mg/dl, NGF-treated; 47,900 ± 3,220 min*mg/dl: *p = 0.0348). NGF treatment of islets was also associated with increased graft failure [the percentage of TdT-mediated dUTP-biotin nick-end labeling (TUNEL)-positive and necrotic transplanted islets on POD 5, control; 23.8% (5/21), NGF-treated; 52.9% (9/17): p = 0.0650] following intraportal islet transplantation. Nonviable (TUNEL-positive and necrotic) islets in both groups expressed vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). On the other hand, viable (TUNEL-negative and not necrotic) islets in both groups did not express VEGF and HIF-1α. In the present study, pre-transplant NGF treatment was associated with impaired survival and angiogenesis of intraportal islet grafts. The effect of NGF on islet transplantation may significantly vary according to the transplant site.

Keywords: Apoptosis, diabetes, intraportal, islet transplantation, nerve growth factor

Introduction

Islet transplantation has become a valuable therapy for type 1 diabetes mellitus. Although the Edmonton protocol introduced various suggestions for the improvement of islet transplantation,1 there is a concern of deteriorating graft function over time.2 In addition, while single-donor islet transplantation success has been achieved,3-5 most centers still rely on multiple donor organs to achieve initial insulin independence.6 A possible explanation for this problem is that many islets are destroyed immediately following transplantation, before a vascular network is re-established.7-12 Although native islets in the pancreas have a rich microvasculature, islet blood vessels are disrupted during islet isolation. Proper revascularization of the transplanted islets is of great importance for the function and survival of islet grafts.

Nerve growth factor (NGF), which is well known as a neurotrophic factor,13,14 has recently been recognized as an angiogenic factor in several tissues15-18 and is reported to play an important regulatory role in pancreatic β-cell function.19-22 We previously demonstrated that treatment of pancreatic islets with NGF improved their quality and viability. Revascularization and survival of islets transplanted under the kidney capsule were improved by NGF.20 Moreover, islets were efficiently transplanted in vascularized chambers based on the neurovascular support from the femoral artery, vein and nerve, in the presence of a gelatin sponge scaffold with NGF.23 However, the usefulness of NGF in intraportal islet transplantation was still unknown. To resolve this problem, we transplanted syngeneic islets (360 islet equivalents per recipient) cultured with or without 2.5S mouse NGF for 24 h into the portal vein of streptozotocin-induced diabetic BALB/c mice.

Results

Islet viability and function test in vitro

At 24 h after culture, islet viability was improved by NGF treatment in a dose-dependent manner (control; 84.0 ± 2.41%, NGF 20 ng/mL; 86.5 ± 2.51%, NGF 100 ng/mL; 91.7 ± 1.55%*, NGF 500 ng/mL; 95.5 ± 0.852%**: *p < 0.05, **p < 0.01 vs. control, ANOVA p = 1.06 × 10−5) (Fig. 1A). However, there were no significant differences between these groups in terms of islet function (Stimulation Index, control; 1.78 ± 0.345, NGF 20 ng/mL; 1.92 ± 0.347, NGF 100 ng/mL; 1.72 ± 0.203, NGF 500 ng/mL; 1.45 ± 0.279: ANOVA p = 0.735) (Fig. 1B).

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Figure 1. Effect of NGF treatment for 24 h in culture on in vitro islet viability and function test. (A) Islet viability was tested by fluorescence microscopy using SYTO green (green for viable area) and ethidium bromide (red for dead area). The ratio of the green area relative to total stained (green + red) area was calculated. It was improved by NGF treatment in a dose-dependent manner (control; 84.0 ± 2.41%, NGF 20 ng/mL; 86.5 ± 2.51%, NGF 100 ng/mL; 91.7 ± 1.55%*, NGF 500 ng/mL; 95.5 ± 0.852%**: *p < 0.05, **p < 0.01 vs. NGF 0 ng/mL, ANOVA p = 1.06 × 10−5). (B) Islet function was tested by glucose-stimulated insulin secretion assay. Stimulation Index was calculated as the ratio of the insulin secretion in the high glucose relative to that in the low glucose. There were no significant differences between these groups (Stimulation Index, control; 1.78 ± 0.345, NGF 20 ng/mL; 1.92 ± 0.347, NGF 100 ng/mL; 1.72 ± 0.203, NGF 500 ng/mL; 1.45 ± 0.279: ANOVA p = 0.735). Data are reported as the mean ± standard error of the mean.

Blood glucose and intraperitoneal glucose tolerance tests (IPGTT)

During the observation period, 44.4% (4/9) of control and 12.5% (1/8) of NGF-treated mice were normal glycemic up to POD 28 (p = 0.195) (Fig. 2). Area under the curve of IPGTT on postoperative days (POD) 30 in the control group was significantly lower than that of the NGF-treated group (control; 35,800 ± 3,960 min*mg/dl, NGF-treated; 47,900 ± 3,220 min*mg/dl: *p = 0.0348) (Fig. 3).

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Figure 2. Effect of pre-transplant NGF treatment on glucose control. Streptozotocin-induced diabetic BALB/c mice received syngeneic islets (360 islet equivalents per recipient) cultured for 24 h with or without 2.5 S mouse NGF (100ng/ml) via the portal vein. Blood glucose levels in the control (A) and NGF-treated (B) groups after transplantation. Bold lines represent parameters of normal glycemic mice in both groups. (C) Achievement of normal glycemia up to POD 28 was defined as a non-fasting blood glucose level consistently maintained ≤ 11 mmol/L (200 mg/dL) after transplantation. During the observation period, 44.4% (4/9) of control and 12.5% (1/8) of NGF-treated mice attained normoglycemia (p = 0.195).

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Figure 3. Results of intraperitoneal glucose tolerance tests (IPGTT) on POD 30. (A) IPGTT were performed by overnight fasting for 12 h and then injecting mice with 2.0 g/kg body weight of glucose solution followed by tail vein blood samples at 0, 15, 30, 60, 90 and 120 min after injection. (B) Area under the curve of IPGTT in the control group was significantly lower than that of the NGF-treated group (control; 35,800 ± 3,960 min*mg/dl, NGF-treated; 47,900 ± 3,220 min*mg/dl: p = 0.0348). Data are reported as the mean ± standard error of the mean.

Histlogical findings for insulin, NGF, TdT-mediated dUTP-biotin nick-end labeling (TUNEL), and hematoxylin and eosin (H&E) 2 h after transplantation

The total number of transplanted islets recovered for histological analysis in the control group was 22 (n = 3) vs. 49 (n = 4) in the NGF-treated group. Most of the transplanted islets were not apoptotic (TdT-mediated dUTP-biotin nick-end labeling (TUNEL)-negative) (control; 20/22, NGF-treated; 46/49: p = 0.651) (Fig. 4C and G), and all of them were not necrotic (Fig. 4D and H) in both groups (Table 1). The ratio of intra-islet NGF positive area to each transplanted islet area was 55.9 ± 6.83% in the control, and that was 77.4 ± 3.73% in the NGF-treated group (*p = 0.0125). Pre-transplant co-culture with NGF significantly increased intra-islet detection of NGF 2 h after transplantation (Fig. 4B, F and I). NGF-positive liver regions were observed in both groups, and most part of those regions corresponded with TUNEL-positive regions. However, NGF and TUNEL-positive liver regions were not necrotic on H&E staining. The proportion of transplanted islets that were in contact with NGF and TUNEL-positive liver regions was 59.1% (13/22) in the control group, and 85.7% (42/49) in the NGF-treated group (*p = 0.0130) (Table 1).

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Figure 4. Histological findings 2 h after transplantation. Insulin staining (A), NGF staining (B), TUNEL (C), and H&E staining (D) were performed in the control group. Insulin staining (E), NGF staining (F), TUNEL (G), and H&E staining (H) were performed in the NGF-treated group. The same islet was used for (A–D) and (E–H). Most of the transplanted islets were TUNEL-negative (not apoptotic) (control; 20/22, NGF-treated; 46/49: p = 0.651) (C and G), and all of them were not necrotic in both groups (D and H) (see Table 1). NGF-positive liver regions were observed in both groups, and most parts of those regions corresponded with TUNEL-positive regions. The ratio of transplanted islets that were in contact with NGF and TUNEL-positive liver regions was 59.1% (13/22) in the control group, and 85.7% (42/49) in the NGF-treated group (*p = 0.0130) (B, C, F and G) (see Table 1). (I) The ratio of intra-islet NGF positive area to each transplanted islet area was 55.9 ± 6.83% in the control, and that was 77.4 ± 3.73% in the NGF-treated group (*p = 0.0125). Pre-transplant co-culture with NGF significantly increased intra-islet detection of NGF 2 h after transplantation. Calibration bar = 100 μm. Data are reported as the mean ± standard error of the mean.

Table 1. Transplanted islet findings at 2 h after intraportal transplantation. Most of the transplanted islets were not TUNEL-positive (apoptotic), and all of them were not necrotic in both groups. More transplanted islets in the NGF group were in contact with NGF and TUNEL-positive liver regions compared with the control group. *Significant difference from the control group (p < 0.05).

2 h after transplantation Control group (n = 22) NGF-treated group (n = 49)
TUNEL positive islets
 9.10%
 6.12%
Necrotic islets
 0.00%
 0.00%
Islets in contact with NGF and TUNEL-positive liver regions 59.1% *85.7%
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Histological findings for insulin, vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1α (HIF-1α), TUNEL, and H&E on POD 5

In the control group, 21 transplanted islets were recovered from five mice. In the NGF-treated group, 17 transplanted islets were recovered from five mice. The percentage of TUNEL-positive islets was 23.8% (5/21) in the control and 52.9% (9/17) in the NGF-treated group (p = 0.0650). TUNEL-positive islets in both groups were necrotic, expressed vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) in all areas, and were in contact with TUNEL-positive, necrotic and HIF-1α positive liver regions (Fig. 5A–E and K–O). Cellular infiltration was observed around the nonviable (TUNEL-positive and necrotic) islets and liver regions in both groups (Fig. 5E and O). On the other hand, TUNEL-negative islets in both groups were not necrotic, did not express VEGF or HIF-1α at all, and the liver parenchyma in the immediate vicinity appeared normal (Fig. 5F–J and P–T).

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Figure 5. Histological findings on POD 5. Insulin staining (A and F), TUNEL (B and G), HIF-1α staining (C and H), VEGF staining (D and I), and H&E staining (E and J) were performed in the control group. Insulin staining (K and P), TUNEL (L and Q), HIF-1α staining (M and R), VEGF staining (N and S), and H&E staining (O and T) were performed in the NGF-treated group. The same islet was used for (A–E), (F–J), (K–O) and (P–T). Nonviable (TUNEL-positive and necrotic in H&E staining) islets in both groups (A–E, K–O) expressed VEGF and HIF-1α in all islet area and were in contact with nonviable and severely hypoxic (HIF-1α positive) liver regions. The percentage of nonviable islets was 23.8% (5/21) in the control (A–E) and 52.9% (9/17) in the NGF-treated group (K–O). Viable (TUNEL-negative and not necrotic in H&E staining) islets in both groups (F–J, P–T) did not express VEGF and HIF-1α at all, and the liver parenchyma in the immediate vicinity appeared normal. The percentage of viable islets was 76.2% (16/21) in the control (F–J) and 47.1% (8/17) in the NGF-treated group (P–T) (p = 0.0650). Cellular infiltration was observed around the dead islets and liver regions in both groups. Arrows indicate cellular infiltration (E and O). Calibration bar = 100 μm.

Blood vessel numbers of viable grafts on POD 5

In the control group, 16 transplanted islets retrieved from five mice were viable. In the NGF-treated group, eight transplanted islets retrieved from five mice were viable. The blood vessel number of the viable grafts was equal between these groups (control; 4.17 ± 0.834 × 10−4/µm2, hepatectomy; 4.34 ± 0.718 × 10−4/µm2: p = 0.886) (Fig. 6A–C).

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Figure 6. Blood vessel numbers of viable grafts on POD 5. Histological staining for CD31 was performed to count the vessel numbers of viable grafts. Islet viability had been confirmed by TUNEL method and H&E staining before vascular assessment. In the control group, 16 transplanted islets were viable from five mice (A). In the NGF-treated group, eight transplanted islets were viable from five mice (B). The blood vessel numbers in viable grafts were equal between these groups (control: 4.17 ± 0.834 × 10−4/µm2; hepatectomy: 4.34 ± 0.718 × 10−4/µm2, p = 0.886) (C). Arrows indicate typical blood vessel morphology in high magnification (A and B). The dotted line is drawn along the margin of the transplanted islets. Calibration bar = 100 μm (low magnification) and 20 μm (high magnification).

Discussion

It has been reported that NGF induces the expression of VEGF for angiogenesis in several tissues15-17 and plays an important regulatory role in pancreatic β-cell function.19-22 However, we observed that NGF-treated islets led to worse graft function following intraportal islet transplantation when compared with untreated islets. Considerably more NGF-treated islets were nonviable (TUNEL-positive and necrotic), and revascularization of viable (TUNEL-negative and not necrotic) grafts was not enhanced several days after transplantation. Islets expressing VEGF in both NGF-treated and control groups appeared severely hypoxic (HIF-1α positive). On the other hand, viable islets did not express VEGF. This might suggest that VEGF is not induced by pre-transplant NGF treatment, but by other stresses related to transplantation.

According to biopsy examination 2 h after transplantation, most of the transplanted islets were TUNEL-negative and all of them were not necrotic in both groups. However, significantly more transplanted islets in the NGF-treated group were in contact with NGF-positive and TUNEL-positive (but not necrotic) liver regions compared with those in the control group. It is known that NGF has cell protective properties in several tissues.24-26 NGF might be upregulated in hepatocytes that were under stress after transplantation. Liver injury might be induced more frequently by NGF-treated islets in several hours after intraportal transplantation. On POD 5, there were more nonviable transplanted islets in the NGF-treated group than in the control group. All of them were in contact with nonviable liver regions, and cellular infiltration was observed around those tissues. Inflammation was frequently induced in the NGF-treated group, and this might be an important reason for graft failure after intraportal transplantation.

One of the important issues for the success of pre-transplant treatment may be the dose of NGF. This study revealed that NGF treatment improved islet viability in vitro in a dose-dependent manner, and we previously used 500 ng/mL of NGF and showed that the revascularization and survival of islets transplanted under the kidney capsule were improved.20 Although 500 ng/mL of NGF was used for this model at first, we had worse results in the NGF group (data not shown). Therefore, we selected a concentration of 100 ng/mL of NGF for pre-transplant treatment and confirmed its effect on intra-islet NGF 2 h after transplantation.

It was previously shown that treatment of pancreatic islets with NGF improved their quality and viability after 96 h in culture.20 In this study, islet viability was improved by 24 h of culture with NGF. However, we found that NGF-treated islets tended to become apoptotic several days after transplantation into the liver. The reason for this unexpected result remains unclear. Several studies have shown that NGF seems to have not just an islet regulatory role19-22 but also pro-inflammatory properties,27-30 mediating effects such as lymphocyte31-35 and macrophage activation,32,36,37 eosinophil chemotaxis,38 and mast cell survival and degranulation.39 One possible explanation for the discrepancy in our results is that NGF-treated islets may stimulate immunocompetent cells after intraportal islet transplantation. The liver has a powerful innate immune system to establish defense mechanisms against potentially toxic antigens. This system comprises a rich complement of innate immune cells, such as macrophages, natural killer cells and natural killer T cells.40,41 It has been revealed that these immune cells played key roles for early graft loss after islet transplantation.42-44 They might be more activated by NGF-treated islets after transplantation and intensively attack these grafts. On the other hand, islets transplanted under the kidney capsule or in vascularized chambers may have less contact with innate immune cells compared with islets transplanted into liver. Therefore, they can avoid early graft loss after transplantation, and their revascularization and survival may be improved by NGF.

Another possible explanation for the discrepancy in the effect of NGF-treatment between intraportal and subcapsular transplants is the dispersion of islets in liver sinusoids. Any NGF effect on inter-islet neurovascular enrichment would be more evident when islets are clustered together under the kidney capsule20 or in biocompatible synthesized chambers.23 This juxtaposition of islets may also explain the higher efficiency of subcapsular transplants.

While NGF may not contribute to intraportal islet transplant outcome, Shimoda et al. reported that VEGF treatment improved that outcome and promoted graft revascularization.45 As described above, several studies suggest that NGF can activate immunocompetent cells such as lymphocytes, macrophages and granulocytes.27-39 Although several reports show the relationship between VEGF and inflammation,46,47 to our knowledge there is no compelling evidence that VEGF directly activates immunocompetent cells. This difference in pro-inflammatory properties of growth factors might be one of the reasons for the discrepancy in transplant outcome. Another possible explanation may be the methodology in treatment with growth factors. We added 2.5 S NGF in the culture medium before transplantation. On the other hand, Shimoda et al.45 delivered VEGF gene into liver immediately after transplantation. This might be another reason for the discrepancy between our results and theirs.

In conclusion, NGF-treated islets have a higher tendency for graft failure after intraportal transplantation. Pre-transplant NGF treatment may impair the survival and angiogenesis of intraportal islet grafts. Thus, the effect of NGF on islet transplantation may be highly dependent on the transplant site.

Materials and Methods

Animals

BALB/c male mice (22–27 g, Charles River) were used as both donors and recipients. The mice were housed under pathogen-free conditions with a 12 h light cycle and free access to food and water. All animal care and treatment procedures were handled in accordance with Principles of Laboratory Animal Care published by the National Institutes of Health (NIH) and approved by the Institutional Animal Care Use Committee.

Induction of diabetes in recipient mice

Streptozotocin (STZ, 200 mg/kg per mouse, Sigma-Aldrich, S0130–1G) was injected intraperitoneally and blood glucose levels were measured by Accu-Chek Aviva glucose monitors (Roche, 03532275004). Mice were transplanted once blood glucose levels were greater than 22 mmol/L (400 mg/dL).

Islet isolation, culture and transplantation. Murine islets were isolated by collagenase (collagenase V, Sigma-Aldrich, C9263–1G) digestion, and separated by Ficoll (Sigma-Aldrich, F4375–500G) discontinuous gradients and purified as previously described.48 Islets were cultured in M199 medium containing 10% fetal bovine serum, with or without 100 ng/mL of mouse 2.5S NGF (BD, 356004) at 37°C in 5% CO2 and humidified air for 24 h before transplantation. Cultured syngeneic islets (360 islet equivalents per recipient) were transplanted via the portal vein into each diabetic mouse.49 One islet equivalent was the islet mass equivalent to a spherical islet of 150 μm in diameter.

Islet viability and function test in vitro

Islet viability and function test were performed after 24 h culture with 0 (control), 20, 100 and 500 ng/mL of mouse 2.5S NGF. Fluorescence microscopy was used to evaluate islet cell viability (tested islet number, control; n = 71, NGF 20 ng/mL; n = 76, NGF 100 ng/mL; n = 100, NGF 500 ng/mL; n = 116). In brief, islets were stained by SYTO green (Invitrogen, S7575) and ethidium bromide (Sigma-Aldrich, E1510–10ML). Images were captured and viable (green) cells and dead (red) cells were manually outlined (ImageJ, NIH) to measure the size. The ratio of the green area relative to total stained (green + red) area was calculated as islet viability. Glucose-stimulated insulin secretion assay was performed to evaluate islet function test (control; n = 9, NGF 20 ng/mL; n = 9, NGF 100 ng/mL; n = 9, NGF 500 ng/mL; n = 9). Aliquots of 20 islet equivalents were washed with low (3.3 mmol/L) glucose medium before glucose challenge. Islets were incubated in low or high (16.5 mmol/L) glucose medium at 37°C in 5% CO2 for 1 h. Supernatant samples were collected for measurement of insulin concentration by a rat/mouse insulin enzyme-linked immunosorbent assay kit (Millipore, EZRMI-13K). The stimulation index was calculated as the ratio of the insulin secretion in the high glucose relative to that in the low glucose.

Islet graft function parameters

Blood glucose was measured at POD 0, 1, 3, 5, 7, 10, 14, 21 and 28. Achievement of normal glycemia up to POD 28 was defined that a non-fasting blood glucose level was consistently maintained ≤ 11 mmol/L (200 mg/dL) after transplantation. Glucose tolerance was assessed on POD 30 (control; n = 9, NGF-treated; n = 8). IPGTT were performed by overnight fasting for 12 h and then injecting mice with 2.0 g/kg body weight of glucose solution followed by tail vein blood sampling at 0, 15, 30, 60, 90 and 120 min after injection. Blood glucose levels were measured by Accu-Chek Aviva glucose monitors.

Histological assessment of insulin, NGF, TUNEL and H&E 2 h after transplantation

Histological assessments were performed in the harvested livers 2 h after transplantation (control; n = 3, NGF-treated; n = 4). The fixed livers were embedded in paraffin and cut in 5 μm thick sections. Staining was performed on three sections from each animal. Specimens were stained by immunohistochemistry for insulin to identify islets and NGF to confirm the effect of the pre-transplant NGF treatment on islets. Primary antibodies were guinea pig anti-insulin antibody (Dako, IR002) diluted 1:100 and rabbit anti-NGF antibody (Santa Cruz Biotechnology Inc., sc-549) diluted 1:100. After incubating with biotinylated secondary Immunoglobulin G antibody (Vector Laboratories, PK-6101), a peroxidase substrate solution containing AEC+ (red for insulin, Dako, K3469) or 3,3′-diaminobenzidine (DAB, brown for NGF, Dako, K3468) was used for visualization and counterstained with hematoxylin.11 Images were captured and intra-islet NGF positive area and transplanted islets were manually outlined with ImageJ to measure the size. The ratio of intra-islet NGF positive area to each transplanted islet area was calculated to evaluate intra-islet detection of NGF. Apoptosis was detected by the TUNEL method using an in situ apoptosis detection kit (Promega, G7130). Sections were treated with proteinase K (Dako, S3020) and incubated with TdT enzyme for 60 min at 37°C. After washing in phosphate buffered saline, the sections were further incubated with streptavidin horseradish peroxidase solution and visualized with DAB.11 H&E staining was performed for detection of necrosis and cellular infiltration. Necrosis was defined as destruction of cell structure with granulation and disappearance of nuclear. Apoptosis and necrosis of transplanted islets were scored as positive or negative.

Histological assessments of insulin, VEGF, HIF-1α, TUNEL, H&E, and CD31 on POD 5

Histological assessments were performed in the harvested livers on POD 5 (control; n = 5, NGF-treated; n = 5). The fixed livers were embedded in paraffin and cut in 5 μm thick sections. Staining was performed on three sections from each animal. Insulin staining, TUNEL method and H&E staining were performed using the same procedure as above. Specimens were stained by immunohistochemistry for VEGF for vascularization50,51 and HIF-1α to determine hypoxia.11,12 Primary antibodies were goat anti-VEGF antibody (Santa Cruz Biotechnology Inc., sc-1836) diluted 1:50, and goat anti-HIF-1α antibody (Santa Cruz Biotechnology Inc., sc-12542) diluted 1:25. After incubating with biotinylated secondary Immunoglobulin G antibody (Vector Laboratories, PK-6105), a peroxidase substrate solution containing DAB (Brown for VEGF and HIF-1α) was used for visualization and counterstained with hematoxylin.11 Apoptosis, necrosis, ischemia and vascularization of transplanted islets were scored as positive or negative. Histological staining for CD31 was performed to count the vessel numbers of viable grafts. These islet viabilities had been confirmed in TUNEL method and H&E staining before CD31 staining. Viable islets were defined as TUNEL-negative and their morphology was normal in H&E staining. The primary antibody was rabbit anti-CD31 antibody (Abcam, ab28364) diluted 1:50. After incubating with biotinylated secondary Immunoglobulin G antibody (Vector Laboratories, PK-6101), a peroxidase substrate solution containing 3,3′-diaminobenzidine (DAB, brown for CD31, Dako) was used for visualization and counterstained with hematoxylin. The blood vessels of grafted islets were identified as CD31 positive zones that were in contact with or within the islets and counted by double-blinded operators at higher magnification (200×). We assessed the number of vessels found per area of each transplanted islet. Images were captured and islet area was manually outlined (ImageJ) to measure the size.

Statistical analyses

All the data are expressed as the mean ± standard error of the mean. Comparisons between the two groups were performed by using Student’s t-test. One-factor ANOVA with Bonferroni-Dunn post hoc test was used to determine the dose-dependent effect of NGF treatment on in vitro islet viability and function test. Fisher’s exact probability test was used for the differences between categorical variables. Analysis of curative rate was performed by Kaplan-Meier method with a log-rank test. Statistical significance was established at p < 0.05.

Acknowledgments

This work was supported by NIH/NIDDK grant number DK077541 (EH). We thank the microsurgical technical support by John Chrisler and the kind help in specimen processing by John Hough.

Glossary

Abbreviations:

NGF

nerve growth factor

TUNEL

TdT-mediated dUTP-biotin nick-end labeling

H&E

hematoxylin and eosin

VEGF

vascular endothelial growth factor

HIF-1α

hypoxia-inducible factor-1α

POD

postoperative days

IPGTT

intraperitoneal glucose tolerance tests

NIH

National Institutes of Health

STZ

streptozotocin

DAB

3,3′-diaminobenzidine

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

References

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