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Nerve growth factor is associated with islet graftfailure following intraportal transplantation

Yukihiko Saito,1,2 Nathaniel K. Chan,1 Naoaki Sakata3 and Eba Hathout1,*

1Islet Transplant Laboratory; Department of Pediatrics; Loma Linda University School of Medicine; Loma Linda, CA USA; 2Division of Advanced Surgical Science and Technology;

Department of Surgery; Tohoku University; Sendai, Japan; 3Division of Hepato-Biliary Pancreatic Surgery; Department of Surgery; Tohoku University; Sendai, Japan

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

Abbreviations: NGF, nerve growth factor; TUNEL, TdT-mediated dUTP-biotin nick-end labeling; H&E, hematoxylin and eosin;VEGF, vascular endothelial growth factor; HIF-1a, hypoxia-inducible factor-1a; POD, postoperative days; IPGTT, intraperitoneal

glucose tolerance tests; NIH, National Institutes of Health; STZ, streptozotocin; DAB, 3,3'-diaminobenzidine

Nerve growth factor (NGF) has recently been recognized as an angiogenic factor with an important regulatory role inpancreatic b-cell function. We previously showed that treatment of pancreatic islets with NGF improved their quality andviability. 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, wetransplanted syngeneic islets (360 islet equivalents per recipient) cultured with or without NGF into the portal veinof streptozotocin-induced diabetic BALB/c mice. Analysis revealed that 44.4% (4/9) of control and 12.5% (1/8) ofNGF-treated mice attained normoglycemia (# 200 mg/dL) (p = 0.195). NGF-treated islets led to worse graft function (areaunder the curve of intraperitoneal glucose tolerance tests (IPGTT) on post-operative day (POD) 30, control; 35,800 ± 3,960min*mg/dl, NGF-treated; 47,900 ± 3,220 min*mg/dl: *p = 0.0348). NGF treatment of islets was also associated withincreased graft failure [the percentage of TdT-mediated dUTP-biotin nick-end labeling (TUNEL)-positive and necrotictransplanted islets on POD 5, control; 23.8% (5/21), NGF-treated; 52.9% (9/17): p = 0.0650] following intraportal islettransplantation. Nonviable (TUNEL-positive and necrotic) islets in both groups expressed vascular endothelial growthfactor (VEGF) and hypoxia-inducible factor-1a (HIF-1a). On the other hand, viable (TUNEL-negative and not necrotic) isletsin both groups did not express VEGF and HIF-1a. In the present study, pre-transplant NGF treatment was associated withimpaired survival and angiogenesis of intraportal islet grafts. The effect of NGF on islet transplantation may significantlyvary according to the transplant site.

Introduction

Islet transplantation has become a valuable therapy for type 1diabetes mellitus. Although the Edmonton protocol introducedvarious suggestions for the improvement of islet transplantation,1

there is a concern of deteriorating graft function over time.2 Inaddition, while single-donor islet transplantation success has beenachieved,3-5 most centers still rely on multiple donor organs toachieve initial insulin independence.6 A possible explanationfor this problem is that many islets are destroyed immediatelyfollowing transplantation, before a vascular network is re-established.7-12 Although native islets in the pancreas have a richmicrovasculature, islet blood vessels are disrupted during isletisolation. Proper revascularization of the transplanted islets is ofgreat importance for the function and survival of islet grafts.

Nerve growth factor (NGF), which is well known as a neuro-trophic factor,13,14 has recently been recognized as an angiogenicfactor in several tissues15-18 and is reported to play an important

regulatory role in pancreatic β-cell function.19-22 We previouslydemonstrated that treatment of pancreatic islets with NGFimproved their quality and viability. Revascularization and survivalof islets transplanted under the kidney capsule were improved byNGF.20 Moreover, islets were efficiently transplanted in vascularizedchambers based on the neurovascular support from the femoralartery, vein and nerve, in the presence of a gelatin sponge scaffoldwith NGF.23 However, the usefulness of NGF in intraportal islettransplantation was still unknown. To resolve this problem, wetransplanted syngeneic islets (360 islet equivalents per recipient)cultured with or without 2.5S mouse NGF for 24 h into the portalvein of streptozotocin-induced diabetic BALB/c mice.

Results

Islet viability and function test in vitro. At 24 h after culture, isletviability was improved by NGF treatment in a dose-dependentmanner (control; 84.0 ± 2.41%, NGF 20 ng/mL; 86.5 ± 2.51%,

*Correspondence to: Eba Hathout; Email: ehathout@llu.eduSubmitted: 09/07/11; Revised: 10/18/11; Accepted: 10/18/11http://dx.doi.org/10.4161/isl.18467

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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 � 1025) (Fig. 1A). However, there were no significantdifferences 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).

Blood glucose and intraperitoneal glucose tolerance tests(IPGTT). During the observation period, 44.4% (4/9) of controland 12.5% (1/8) of NGF-treated mice were normal glycemic up toPOD 28 (p = 0.195) (Fig. 2). Area under the curve of IPGTT onpostoperative days (POD) 30 in the control group was significantlylower 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).

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 trans-planted islets recovered for histological analysis in the controlgroup was 22 (n = 3) vs. 49 (n = 4) in the NGF-treated group.Most of the transplanted islets were not apoptotic (TdT-mediateddUTP-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 eachtransplanted islet area was 55.9 ± 6.83% in the control, and thatwas 77.4 ± 3.73% in the NGF-treated group (*p = 0.0125). Pre-transplant co-culture with NGF significantly increased intra-isletdetection of NGF 2 h after transplantation (Fig. 4B, F and I).NGF-positive liver regions were observed in both groups, andmost part of those regions corresponded with TUNEL-positiveregions. However, NGF and TUNEL-positive liver regions werenot necrotic on H&E staining. The proportion of transplantedislets that were in contact with NGF and TUNEL-positive liverregions was 59.1% (13/22) in the control group, and 85.7%(42/49) in the NGF-treated group (*p = 0.0130) (Table 1).

Histological findings for insulin, vascular endothelial growthfactor (VEGF), hypoxia-inducible factor-1a (HIF-1a), TUNEL,and H&E on POD 5. In the control group, 21 transplanted isletswere recovered from five mice. In the NGF-treated group, 17transplanted islets were recovered from five mice. The percentage ofTUNEL-positive islets was 23.8% (5/21) in the control and 52.9%(9/17) in the NGF-treated group (p = 0.0650). TUNEL-positiveislets in both groups were necrotic, expressed vascular endothelialgrowth factor (VEGF) and hypoxia-inducible factor-1a (HIF-1a)in all areas, and were in contact with TUNEL-positive, necrotic and

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 microscopyusing 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) areawas 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 � 1025). (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 thelow 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.

RESEARCH PAPER

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Do not distribute.HIF-1a positive liver regions (Fig. 5A–E and K–O). Cellularinfiltration was observed around the nonviable (TUNEL-positiveand necrotic) islets and liver regions in both groups (Fig. 5E andO). On the other hand, TUNEL-negative islets in both groupswere not necrotic, did not express VEGF or HIF-1a at all, and theliver parenchyma in the immediate vicinity appeared normal(Fig. 5F–J and P–T).

Blood vessel numbers of viable grafts on POD 5. In thecontrol group, 16 transplanted islets retrieved from five mice wereviable. In the NGF-treated group, eight transplanted isletsretrieved from five mice were viable. The blood vessel numberof the viable grafts was equal between these groups (control;4.17 ± 0.834 � 1024/mm2, hepatectomy; 4.34 ± 0.718 � 1024/mm2: p = 0.886) (Fig. 6A–C).

Discussion

It has been reported that NGF induces the expression of VEGFfor angiogenesis in several tissues15-17 and plays an importantregulatory role in pancreatic β-cell function.19-22 However, weobserved that NGF-treated islets led to worse graft functionfollowing intraportal islet transplantation when compared withuntreated islets. Considerably more NGF-treated islets werenonviable (TUNEL-positive and necrotic), and revascularizationof viable (TUNEL-negative and not necrotic) grafts was notenhanced several days after transplantation. Islets expressingVEGF in both NGF-treated and control groups appeared severelyhypoxic (HIF-1a positive). On the other hand, viable islets did

Figure 2. Effect of pre-transplant NGF treatment on glucose control. Streptozotocin-induced diabetic BALB/c mice received syngeneic islets (360 isletequivalents 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) andNGF-treated (B) groups after transplantation. Bold lines represent parameters of normal glycemic mice in both groups. (C) Achievement of normalglycemia up to POD 28 was defined as a non-fasting blood glucose level consistently maintained# 11 mmol/L (200 mg/dL) after transplantation. Duringthe observation period, 44.4% (4/9) of control and 12.5% (1/8) of NGF-treated mice attained normoglycemia (p = 0.195).

Figure 3. Results of intraperitoneal glucose tolerance tests (IPGTT) onPOD 30. (A) IPGTT were performed by overnight fasting for 12 h and theninjecting mice with 2.0 g/kg body weight of glucose solution followedby 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 wassignificantly 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.

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Do not distribute.not express VEGF. This might suggest that VEGF is not inducedby pre-transplant NGF treatment, but by other stresses related totransplantation.

According to biopsy examination 2 h after transplantation,most of the transplanted islets were TUNEL-negative and all ofthem were not necrotic in both groups. However, significantlymore transplanted islets in the NGF-treated group were in contactwith NGF-positive and TUNEL-positive (but not necrotic) liverregions compared with those in the control group. It is knownthat NGF has cell protective properties in several tissues.24-26 NGF

might be upregulated in hepatocytes that were under stress aftertransplantation. Liver injury might be induced more frequently byNGF-treated islets in several hours after intraportal transplanta-tion. On POD 5, there were more nonviable transplanted islets inthe NGF-treated group than in the control group. All of themwere in contact with nonviable liver regions, and cellular infiltra-tion was observed around those tissues. Inflammation was fre-quently induced in the NGF-treated group, and this might be animportant reason for graft failure after intraportal transplantation.

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

It was previously shown that treatment of pancreatic islets withNGF improved their quality and viability after 96 h in culture.20

In this study, islet viability was improved by 24 h of culture withNGF. However, we found that NGF-treated islets tended to

Figure 4. Histological findings 2 h after transplantation. Insulin staining (A), NGF staining (B), TUNEL (C), and H&E staining (D) were performed in thecontrol 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 usedfor (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 allof 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 thoseregions corresponded with TUNEL-positive regions. The ratio of transplanted islets that were in contact with NGF and TUNEL-positive liver regions was59.1% (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 NGFpositive 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 mm. Data arereported 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 allof them were not necrotic in both groups. More transplanted islets in theNGF group were in contact with NGF and TUNEL-positive liver regionscompared with the control group. *Significant difference from the controlgroup (p , 0.05)

2 h after transplantationControl 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 andTUNEL-positive liver regions

59.1% *85.7%

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Do not distribute.become apoptotic several days after transplantation into the liver.The reason for this unexpected result remains unclear. Severalstudies have shown that NGF seems to have not just an isletregulatory role19-22 but also pro-inflammatory properties,27-30

mediating effects such as lymphocyte31-35 and macrophageactivation,32,36,37 eosinophil chemotaxis,38 and mast cell survivaland degranulation.39 One possible explanation for the discrepancyin our results is that NGF-treated islets may stimulate immuno-competent cells after intraportal islet transplantation. The liver hasa powerful innate immune system to establish defense mechan-isms against potentially toxic antigens. This system comprises arich complement of innate immune cells, such as macrophages,natural killer cells and natural killer T cells.40,41 It has beenrevealed that these immune cells played key roles for early graftloss after islet transplantation.42-44 They might be more activatedby NGF-treated islets after transplantation and intensively attackthese grafts. On the other hand, islets transplanted under thekidney capsule or in vascularized chambers may have less contactwith innate immune cells compared with islets transplantedinto liver. Therefore, they can avoid early graft loss after trans-plantation, and their revascularization and survival may beimproved by NGF.

Another possible explanation for the discrepancy in the effectof NGF-treatment between intraportal and subcapsular trans-plants is the dispersion of islets in liver sinusoids. Any NGF effect

on inter-islet neurovascular enrichment would be more evidentwhen islets are clustered together under the kidney capsule20 or inbiocompatible synthesized chambers.23 This juxtaposition of isletsmay also explain the higher efficiency of subcapsular transplants.

While NGF may not contribute to intraportal islet transplantoutcome, Shimoda et al. reported that VEGF treatment impro-ved that outcome and promoted graft revascularization.45 Asdescribed above, several studies suggest that NGF can activateimmunocompetent cells such as lymphocytes, macrophages andgranulocytes.27-39 Although several reports show the relationshipbetween VEGF and inflammation,46,47 to our knowledge there isno compelling evidence that VEGF directly activates immuno-competent cells. This difference in pro-inflammatory properties ofgrowth factors might be one of the reasons for the discrepancy intransplant outcome. Another possible explanation may be themethodology in treatment with growth factors. We added 2.5 SNGF in the culture medium before transplantation. On the otherhand, Shimoda et al.45 delivered VEGF gene into liver immedi-ately after transplantation. This might be another reason for thediscrepancy between our results and theirs.

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

Figure 5. Histological findings on POD 5. Insulin staining (A and F), TUNEL (B and G), HIF-1a 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-1a staining (M and R), VEGF staining (N and S), and H&Estaining (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 andnecrotic in H&E staining) islets in both groups (A–E, K–O) expressed VEGF and HIF-1a in all islet area andwere in contact with nonviable and severely hypoxic(HIF-1a 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-1a at all, and the liver parenchyma inthe 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 mm.

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Do not distribute.Materials and Methods

Animals. BALB/c male mice (22–27 g, Charles River) were usedas both donors and recipients. The mice were housed underpathogen-free conditions with a 12 h light cycle and free access tofood and water. All animal care and treatment procedures werehandled in accordance with Principles of Laboratory Animal Carepublished by the National Institutes of Health (NIH) andapproved 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 injectedintraperitoneally and blood glucose levels were measured byAccu-Chek Aviva glucose monitors (Roche, 03532275004). Micewere transplanted once blood glucose levels were greater than22 mmol/L (400 mg/dL).

Islet isolation, culture and transplantation. Murine islets wereisolated by collagenase (collagenase V, Sigma-Aldrich, C9263–1G) digestion, and separated by Ficoll (Sigma-Aldrich, F4375–500G) discontinuous gradients and purified as previouslydescribed.48 Islets were cultured in M199 medium containing10% fetal bovine serum, with or without 100 ng/mL of mouse2.5S NGF (BD, 356004) at 37°C in 5% CO2 and humidified airfor 24 h before transplantation. Cultured syngeneic islets (360islet equivalents per recipient) were transplanted via the portalvein into each diabetic mouse.49 One islet equivalent was the isletmass equivalent to a spherical islet of 150 mm in diameter.

Islet viability and function test in vitro. Islet viability and func-tion test were performed after 24 h culture with 0 (control), 20,

100 and 500 ng/mL of mouse 2.5S NGF. Fluorescence micro-scopy 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 stainedby 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 tototal stained (green + red) area was calculated as islet viability.Glucose-stimulated insulin secretion assay was performed toevaluate 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 of20 islet equivalents were washed with low (3.3 mmol/L) glucosemedium before glucose challenge. Islets were incubated in low orhigh (16.5 mmol/L) glucose medium at 37°C in 5% CO2 for 1 h.Supernatant samples were collected for measurement of insulinconcentration by a rat/mouse insulin enzyme-linked immuno-sorbent assay kit (Millipore, EZRMI-13K). The stimulation indexwas calculated as the ratio of the insulin secretion in the highglucose relative to that in the low glucose.

Islet graft function parameters. Blood glucose was measuredat POD 0, 1, 3, 5, 7, 10, 14, 21 and 28. Achievement ofnormal glycemia up to POD 28 was defined that a non-fastingblood glucose level was consistently maintained # 11 mmol/L(200 mg/dL) after transplantation. Glucose tolerance was assessedon POD 30 (control; n = 9, NGF-treated; n = 8). IPGTT wereperformed by overnight fasting for 12 h and then injecting micewith 2.0 g/kg body weight of glucose solution followed by tail

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. Isletviability had been confirmed by TUNEL method and H&E staining before vascular assessment. In the control group, 16 transplanted islets were viablefrom five mice (A). In the NGF-treated group, eight transplanted islets were viable from five mice (B). The blood vessel numbers in viable grafts wereequal between these groups (control: 4.17 ± 0.834 � 1024/mm2; hepatectomy: 4.34 ± 0.718 � 1024/mm2, p = 0.886) (C). Arrows indicate typical bloodvessel morphology in high magnification (A and B). The dotted line is drawn along the margin of the transplanted islets. Calibration bar = 100 mm (lowmagnification) and 20 mm (high magnification).

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vein blood sampling at 0, 15, 30, 60, 90 and 120 min afterinjection. Blood glucose levels were measured by Accu-ChekAviva glucose monitors.

Histological assessment of insulin, NGF, TUNEL and H&E2 h after transplantation. Histological assessments were per-formed in the harvested livers 2 h after transplantation (control;n = 3, NGF-treated; n = 4). The fixed livers were embedded inparaffin and cut in 5 mm thick sections. Staining was performedon three sections from each animal. Specimens were stained byimmunohistochemistry for insulin to identify islets and NGFto confirm the effect of the pre-transplant NGF treatment onislets. 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. Afterincubating with biotinylated secondary Immunoglobulin Gantibody (Vector Laboratories, PK-6101), a peroxidase substratesolution containing AEC+ (red for insulin, Dako, K3469) or 3,3'-diaminobenzidine (DAB, brown for NGF, Dako, K3468) wasused for visualization and counterstained with hematoxylin.11

Images were captured and intra-islet NGF positive area andtransplanted islets were manually outlined with ImageJ to measurethe size. The ratio of intra-islet NGF positive area to each trans-planted islet area was calculated to evaluate intra-islet detection ofNGF. Apoptosis was detected by the TUNEL method using anin situ apoptosis detection kit (Promega, G7130). Sections weretreated with proteinase K (Dako, S3020) and incubated withTdT enzyme for 60 min at 37°C. After washing in phosphatebuffered saline, the sections were further incubated withstreptavidin horseradish peroxidase solution and visualized withDAB.11 H&E staining was performed for detection of necrosisand cellular infiltration. Necrosis was defined as destruction ofcell structure with granulation and disappearance of nuclear.Apoptosis and necrosis of transplanted islets were scored aspositive or negative.

Histological assessments of insulin, VEGF, HIF-1a, TUNEL,H&E, and CD31 on POD 5. Histological assessments wereperformed in the harvested livers on POD 5 (control; n = 5,NGF-treated; n = 5). The fixed livers were embedded in paraffinand cut in 5 mm thick sections. Staining was performed on threesections from each animal. Insulin staining, TUNEL method andH&E staining were performed using the same procedure as above.Specimens were stained by immunohistochemistry for VEGFfor vascularization50,51 and HIF-1a to determine hypoxia.11,12

Primary antibodies were goat anti-VEGF antibody (Santa CruzBiotechnology Inc., sc-1836) diluted 1:50, and goat anti-HIF-1aantibody (Santa Cruz Biotechnology Inc., sc-12542) diluted 1:25.After incubating with biotinylated secondary Immunoglobulin Gantibody (Vector Laboratories, PK-6105), a peroxidase substratesolution containing DAB (Brown for VEGF and HIF-1a) wasused for visualization and counterstained with hematoxylin.11

Apoptosis, necrosis, ischemia and vascularization of transplantedislets were scored as positive or negative. Histological stainingfor CD31 was performed to count the vessel numbers of viablegrafts. These islet viabilities had been confirmed in TUNELmethod and H&E staining before CD31 staining. Viable isletswere defined as TUNEL-negative and their morphology wasnormal in H&E staining. The primary antibody was rabbit anti-CD31 antibody (Abcam, ab28364) diluted 1:50. After incubatingwith biotinylated secondary Immunoglobulin G antibody (VectorLaboratories, PK-6101), a peroxidase substrate solution contain-ing 3,3'-diaminobenzidine (DAB, brown for CD31, Dako) wasused for visualization and counterstained with hematoxylin. Theblood vessels of grafted islets were identified as CD31 positivezones that were in contact with or within the islets and counted bydouble-blinded operators at higher magnification (200�). Weassessed the number of vessels found per area of each transplantedislet. 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 groupswere performed by using Student’s t-test. One-factor ANOVAwith Bonferroni-Dunn post hoc test was used to determine thedose-dependent effect of NGF treatment on in vitro islet viabilityand function test. Fisher’s exact probability test was used for thedifferences between categorical variables. Analysis of curative ratewas performed by Kaplan-Meier method with a log-rank test.Statistical significance was established at p , 0.05.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by NIH/NIDDK grant numberDK077541 (EH). We thank the microsurgical technical supportby John Chrisler and the kind help in specimen processing byJohn Hough.

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