Sustained Reversal of Diabetes Following Islet Transplantation to Striated Musculature in the Rat

Journal of Surgical Research 160, 145–154 (2010) doi:10.1016/j.jss.2008.11.009

Sustained Reversal of Diabetes Following Islet Transplantation to Striated Musculature in the Rat Tormod Lund, M.D.,*,†,1 Olle Korsgren, M.D., Ph.D.,‡ Ingrid A. Aursnes, MSc.,† Hanne Scholz, MSc., Ph.D.,† and Aksel Foss, M.D., Ph.D.*,† *Surgical Clinic, Section for Transplantation; †Institute for Surgical Research, Rikshospitalet University Hospital, Oslo, Norway; and ‡Department of Clinical Immunology, Rudbeck Laboratory, Uppsala University Hospital, Uppsala, Sweden Submitted for publication July 17, 2008

Background. There is an increasing emphasis in the islet transplant community on the development of alternative sites for islet implantation. Striated musculature constitutes a potential alternative, which has been successfully employed in autotransplantation of parathyroid glands for decades. In the present study, a technique for intramuscular islet transplantation was developed and compared with intraportal islet transplantation in a syngeneic rat model. Materials and methods. Lewis rats were used. Pancreata were digested using Liberase. Islets were either transplanted into m. biceps femoris in a pearls-on-astring fashion or intraportally, and the ability to reverse diabetes was compared. Eight weeks after transplantation an IVGTT was performed. Real-time quantitative RT-PCR was employed on muscle biopsies to investigate mRNA levels of cytokines in response to the transplant procedure. Explanted livers, muscles, and pancreata were harvested at the end of the experiment for histopathological analyses. Results. 2000 IEQ repeatedly cured diabetic rats at the intraportal site, while 4000 IEQ was required at the intramuscular site. Time to reversal of diabetes, post-transplant weight development, and IVGTT curves did not differ between the groups. Normoglycemia was sustainable to the end of the study (>100 days) for all animals. The transplant procedure upregulated pro-inflammatory cytokines (IL-6 and IL-8) in striated muscle, and peri-islet fibrosis was observed in intramuscular grafts. Conclusions. Islet transplantation into striated musculature is feasible; however, in its present form

1 To whom correspondence and reprint requests should be addressed at Surgical Clinic, Section for Transplantation, Rikshospitalet University Hospital, N-0027 Oslo, Norway. E-mail: tormod.lund@ rikshospitalet.no.

the intramuscular site is less efficient compared with the liver in rats. The intramuscular site allows manipulation of the graft and implantation site prior to transplantation and may therefore have implications for islet transplantation in humans. Ó 2010 Elsevier Inc. All rights reserved.

Key Words: alternative site; IL-6; intramuscular; intraportal; islet transplantation; IVGTT; rat.

INTRODUCTION

Islet transplantation is being explored as treatment for patients with type 1 diabetes. The short-term efficacy of the procedure is excellent; however, over time most of the recipients have to resume insulin therapy [1]. In patients where insulin independence is achieved, it has been shown that insulin secreting capacity is only 20% to 40% of a healthy person [2, 3], in spite of infusing islets from multiple donors [1]. Intraportal transplantation (IP) of islets is the most common method for islet allo- and autotransplantation [1, 4]. However, functional and experimental studies suggest that a large part of the intraportal (IP) transplanted islets are destroyed shortly after infusion, due to islet–blood interactions [2, 3, 5–8]. In addition, it has been suggested that intrahepatic islets are exposed to high concentrations of diabetogenic immunosuppressive drugs, nutrients, and gut hormones [9]. This may lead to hypersecretion of undiluted insulin into surrounding hepatocytes and focal steatosis [10]. Steatosis following islet transplantation may cause islet lipotoxicity [11] and eventually be a risk factor for the development of adenomas in the liver [12]. Furthermore, when grafted, the vascular density of intrahepatic islets is

145

0022-4804/08 $36.00 Ó 2010 Elsevier Inc. All rights reserved.

146

JOURNAL OF SURGICAL RESEARCH: VOL. 160, NO. 1, MAY 1, 2010

reduced compared with native islets located in the pancreas [13]. Finally, due to procedural risks, it has been difficult to obtain serial biopsies necessary for characterization of islet engraftment or rejection in the liver. For these reasons, development of an alternative site for islet transplantation has been suggested to be an important factor for further progress in the field [14]. Kemp and coworkers concluded some 30 years ago that the liver was the optimal site for islet transplantation in rats [15], but more recent studies have shown superior long term results at other implantation sites, such as beneath the kidney capsule [16, 17] or into the spleen [18, 19]. We hypothesize that an optimal transplant site should minimize direct islet-blood interactions. It should be easy accessible for preconditioning and modulation prior to transplantation. It should be well vascularised to promote engraftment, and it should be possible to excise if necessary. An available, well vascularised transplantation site is striated musculature, which has been employed successfully in autotransplantation of parathyroid tissue for years [20]. Similarities between the parathyroid tissue and pancreatic islets, as well as a few experimental and clinical observations, could indicate that the intramuscular (IM) site is a feasible site for islet transplantation [21–24]. Transplantation of islets to the muscular interstitium would avoid intravascular transplantation and minimize islet–blood contact. The transplantation site would be easy to access and would allow application of bioengineered matrices carrying molecules which potentially could improve islet engraftment and revascularization. The islet mass of an adult human weighs <1 g and is easily soluble in <5 mL of transplant medium. With an unlimited islet source, large amounts of islets could repeatedly be transplanted IM to a preconditioned site. The primary objective of this study was to develop a simple and reproducible IM islet transplantation model in the rat. Secondary objectives were to explore the quantity of islets necessary to obtain insulin independence and to investigate the temporal islet function post-transplant in comparison with the well established IP islet transplantation model. MATERIALS AND METHODS The animal procedures and housing were in accordance with institutional guidelines and national legislation conforming to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication no. 85-23, revised 1996). All animal experiments were approved by the Institutional Animal Care Committee. Syngeneic Lewis rats (Tyconic, Ry, Denmark), age 6 to 8 wk, weighing 250 to 350 g were used. For all procedures, the animals were anesthetized with a subcutaneous injection of a mixture consisting of fentanyl (0.315 mg/mL), fluanisone (10 mg/mL) (Hypnorm,; VetaPharma Ltd., Leeds, UK), and midazolam (5 mg/mL) (Dormicum; Roche, Basel, Switzerland), diluted 1:1:2 in 0.9% NaCl, at a dose of 1.3 mL/kg of body weight.

Induction of Diabetes Diabetes was induced by a single intraperitoneal injection of streptozotocin (65 mg/kg body weight) (Sigma-Aldrich, Steinheim, Germany). Diabetes was defined as two consecutive, nonfasting measurements of blood glucose >20 mmol/L (>360mg/dL) (Ascensia Contour; Bayer HealthCare AG, Leverkusen, Germany) [25].

Isolation of Islets Islets were isolated as previously described [26], with slight modifications. Briefly, the animal was anesthetized with a s.c. injection of hypnorm/dormicum. A middle abdominal incision was subsequently performed. Following occlusion of the pancreatic duct near the duodenum, the common bile duct was cannulated and perfused with 10 mL Liberase solution (0.33 mg/mL; Roche, Indianapolis, IN) in cold Hanks balanced salt solution (HBSS; Sigma, St. Louis, MO). Thereafter a swift thoracotomy was performed with excision of the donor heart, followed by excision of the distended pancreas and placement of the pancreas in a conical tube containing 5 mL of the cold Liberase solution. The cold ischemic time (4 C) was approximately 20 min per pancreas. The resected pancreas was then incubated statically in a 37 C water bath for 20 min. After washing three times with Hanks solution supplemented with 10% fetal calf serum, the tissue pellet was resuspended in cold University of Wisconsin (UW) solution and kept on ice for 30 to 60 min. Storage of the pancreatic digest in UW solution prior to purification has been suggested to improve the islet yield [27]. Islet purification was achieved by a three-layer discontinuous density gradient. Ficoll 1.100 and 1.077 g/mL (Biochrom, Berlin, Germany) were prediluted with Hanks solution to 1.090 and 1.040 g/mL, respectively. The tissue pellet was resuspended in 1.090 g/mL Ficoll and placed at the bottom. Ficoll 1.077 mg/mL was overlaid onto the bottom layer and capped with Ficoll 1.040 g/mL. After centrifugation at 2500 rpm/min for 15 min, islets were harvested from the interface between the 1.077 and 1.040 g/mL layer. Islet preparations were maintained in culture medium (RPMI; ICN Biomedicals, Costa Mesa, CA) supplemented with HEPES, L-glutamine, gentamicin, fungizone (Gibco, Invitrogen, Paisley, Scotland, UK), ciprofloxacin (Bayer Healthcare, Leverkusen, Germany), and 10% fetal calf serum (Invitrogen, CA), at 37 C (5% CO2) overnight. The volume and purity were determined by microscopic sizing after staining with dithizone (Sigma-Aldrich, St. Louis, MO). Islet number were counted and converted to the standard number of islets equivalents (IEQ; diameter standardizing to 150 mm).

Development of the IM Islet Transplantation Model In the primary experiments an open transplantation technique was used. The skin and the fascia over the m. biceps femoris (thigh muscle) were opened and a cavity in the musculature was preformed using careful blunt dissection to avoid bleeding. Islets dispersed in 0.05– 0.2 mL transplant medium were allowed to sink into the muscular cavity while the muscular edges were elevated with stay sutures. Thereafter, the fascia was sutured with 9-0 prolene before skin closure. The procedure was meticulously performed to avoid increment in the intramuscular pressure. None of these transplantations induced sustained reversal of diabetes. Several attempts with traditional IM injection with different volumes of islets were also discouraging, until partial effect was obtained by applying tiny volumes of islets at different sites in the musculature. After numerous testing of different cannulae, transplant volumes, number of islets, and injection techniques, a robust, simple, and reproducible IM islet transplantation model able to persistently reverse diabetes was achieved using the following technique: an i.v. cannula (BD neoflon, 0.7 3 19 mm) was inserted distally in the fiber direction of the m. biceps femoris and mobilized through the muscle to the proximal part. Islets (1500–2000 IEQ) were dispersed in 0.15 mL transplant medium (human serum albumin (50 mg/mL) (Octapharma AG, Ziegelbru¨cke, Switzerland) in ringer-acetate (Fresenius Kabi AB, Uppsala, Sweden)

LUND ET AL.: INTRAMUSCULAR ISLET TRANSPLANTATION supplemented with 5% glucose) and collected in a 1mL syringe. Islets were then carefully injected while slowly moving the cannula from proximal to distal part of the muscle to obtain a pearls-on-a-string distribution of islets in the muscle (Fig. 1). The procedure was repeated to the m. biceps femoris on other extremity.

147

was collected for determination of insulin [measured by EIA (Mercodia AB, Uppsala, Sweden)]. At the end of the study, pancreata from transplanted animals were harvested and assessed for insulin content to confirm graft potency (see below).

PEGylation of Islets Comparative Study of IM- Versus IP Islet Transplantation in a Syngeneic Rat Model After a reproducible model for IM islet transplantation was established, 25 rats were chemically rendered diabetic and the number of islets required to reverse diabetes in the IM and IP groups were determined. Blood sugar levels in diabetic rats were monitored for 7 to 10 d prior to inclusion, to verify persistent levels >20 mmol/L, as potential for endogenous beta-cell neogenesis in this time-period has been suggested [28, 29]. Cure from diabetes was defined as >3 consecutive measurements of blood glucose below 10 mmol/L, partial graft function as blood glucose levels between 10 and 20 mmol/L, and graft non-function as persistent blood glucose levels above 20 mmol/L. The animals were transplanted in parallel with pooled islets to minimize inter-isolation variability. Twelve rats, cured from diabetes, 6 with IP islet transplantation (IPtx) and 6 with IM islet transplantation (IMtx), were used for the long-term comparative study. Four rats served as controls. Three rats served as diabetic nontreated controls. The IPtx group was transplanted intraportally through the coronary branch of the portal vein as described by Jahr and co-workers [26].

Assessment of Graft Function Blood glucose was measured daily in the recipients, until >3 consecutive measurements were below 10 mmol/L. After that, blood glucose was measured once a week. Eight weeks after transplantation, graft function was assessed in four animals in each group by an intravenous glucose tolerance test (IVGTT): Under fasting condition (>6 h), after hypnorm/dormicum induced anesthesia, a catheter was placed in the left v. jugularis externa and 0.5 g/kg b.w. glucose was slowly injected over 30 s. Blood glucose was measured before and 1, 5, 10, 15, 30, 45, 60, and 90 min after injection using a glucometer (see above). The meter displayed a ‘‘Hi’’ when the glucose concentration exceeded 33.3 mmol/L (600 mg/dL); in these cases 33.3 mmol/L was used as value. At time points 0, 5, 10, 15, 30, 60, and 90 min, 0.2 mL blood

FIG. 1. Illustration of the intramuscular transplant technique. (A) An i.v. cannula was inserted distally in the fiber direction of the m. biceps femoris and mobilized through the muscle to the proximal part. (B) Islets were then carefully injected while slowly moving the cannula from proximal to distal part of the muscle to obtain a pearls-on-a-string distribution of islets in the muscle.

PEGylation of islets were performed as described by Panza and coworkers [30]. Briefly, purified islets were pelleted in a microcentrifuge tube at 5000 3 g for 5 s. The supernatant was removed and the cells resuspended in 0.5 mL of 11 mM glucose solution in PBS. The cell suspension was used to solubilize 100 mg of PEG-isocyanate (MW 5000; Nanocs Inc., New York, NY) resulting in a 20% PEG solution. After the PEG-isocyanate was solubilized by the cell suspension, the cells were incubated at 37 C in a humidifed 5% CO2 incubator for 30 minutes with occasional agitation. The islets were pelleted again and the supernatant removed. After PEG-treatment, the islets were transferred to culture medium and maintained overnight.

Immunohistochemical Analyses Explanted muscles, livers, and pancreata were collected in 10% formalin buffer at the end of the experiment. After fixation, the tissue was embedded in paraffin blocks. Tissue sections (5 mm thick) were stained with haematoxylin-eosin and the grafts assessed morphologically. In order to identify islets, insulin and glucagon content was assessed. Primary antibodies and dilutions were as follows: insulin, rabbit (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and glucagon, rabbit (1:500; Chemicon International, Temecula, CA). Avidinbiotincomplexes (ABC) with peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). To detect fibrosis, acid fuchsin orange g (AFOG) staining was performed according to the manufacturer’s description (Merck KGaA, Darmstadt, Germany). Representative slides were evaluated blindly by a pathologist.

Myocyte mRNA Analyses For quantification of muscle inflammatory response to the transplant procedure, a total of 9 Lewis rats were used. The rats were divided into three groups. Group (1) received injection of low-volume transplant medium (0.15 mL, sham), group (2) received injection of high-volume transplant medium (0.60 mL, sham) and, group (3) received 2000 IEQ dispersed in 0.15 mL transplant medium (Tx group). The IM transplant procedure was carried out in all groups, under hypnorm/dormicum anesthesia, according to the described method (see above) on the right m. femoris biceps, while the left m. femoris biceps served as control. One hour after initiation of the procedure, muscle biopsies (50–-60 mg) were harvested from the right and left m. femoris biceps and immediately frozen in liquid nitrogen. The biopsies were subsequently stored at –80 C. For RNA extraction, the tissue was disrupted and homogenized using mortar and pestle, subsequently solubilized in lysis buffer (Qiagen GmbH, Hilden, Germany), followed by three cycles in a rotorstator homogenizer (Ultra-Turraxx; IKA Werke GmbH, Staufen, Germany). RNA was then extracted according to the manufacturer’s protocol (RNeasy; Qiagen GmbH, Hilden, Germany). RNA concentration was determined with a spectrophotometer (NanoDrop ND-1000; NanoDrop Technologies, Inc., Rockland, DE) and stored at –80 C. Quantitative real time RT-PCR was used to determine the expression of IL-6, IL-8, TNFa, and IL-1b mRNA relative to GAPDH as the housekeeping gene. Total RNA (100 ng) from each muscle sample was reverse-transcribed using the TaqMan PCR Core Reagent Kit (Applied Biosystems, Foster City, CA). Each cDNA sample was run three times in duplicates using the ABI Prism 7900 Sequence Detection System and software according to the manufacturer’s instructions (Applied Biosystems). SyBr green assays were performed using 2 3 SyBr Green Universal Master Mix (Applied Biosystems) and 300 nM sense and anti-sense oligonucleotide primers. The specificity of the SyBr Green assays was assessed by melting point analysis. A standard curve was generated by

148

JOURNAL OF SURGICAL RESEARCH: VOL. 160, NO. 1, MAY 1, 2010

amplifications of cDNA obtained from serial dilutions of muscle total RNA. For all specific mRNA amplified linear inverse correlations were observed between amount of RNA and CT value (number of cycles at threshold lines). The primer sequences were as follows for GAPDH: forward 5’-CCAAGGTCATCCATGACAACTT-3’, reverse 5’-AGGGGCCATCCACAGTCTT-3’, IL-8: forward 5’-CACTTCAA GAACATCCAGAGTTTGA-3’, reverse 5’-CCATTCTTGAGTGTGGC TATGACT-3’, IL-6: forward 5’-ATGAAGTTTCTCTCCGCAAGA-3’, reverse 5’CTCCGGACTTGTGAAGTAGGG-3’, TNFa: forward 5’-CTGGGCAGC GTTTATTC-3’, reverse 5’-TTGCTTCTTCCCTGTTCC-3’, and IL-1b: forward 5’-TGTGATGAAAGACGGCACAC-3’, reverse 5’-CTTCTTCTTTG GGTATTGTTTGG-3’.

Statistical Analyses Data are presented as mean 6 SD. Statistical significance was determined using one-way analysis of variance (ANOVA) with post hoc analysis using Tukey’s comparisons. For comparison of two groups, a two-tailed t-test was used. From the IVGTT, the area under the insulin curve (AUCinsulin) was calculated using the trapezoid rule for insulin data from 0 to 90 min. The glucose tolerance was quantified from the glucose elimination constant (KG; expressed as percent elimination of glucose per minute) as the reduction in circulating glucose between 5 and 30 min after intravenous administration following logarithmic transformation of the individual plasma glucose values. A similar estimation was performed for the total 1- to 90-min glucose disappearance rate [KG (1–90)]. Probability values were considered significant at a level of P < 0.05.

RESULTS Determination of Islet Equivalents Necessary to Reverse Diabetes

The number of islets required to cure diabetic rats was determined by progressively increasing the number of transplanted islets from 1000 to 2000 to 3000 and finally 4000 IEQ per transplant at the IP- and IM site. In our transplant models we found that 2000 IEQ cured all recipients at the IP site. All recipients transplanted with 4000 IEQ obtained sustained normoglycemia following IM transplantation (Table 1).

days (range 5–21 d, n ¼ 6). For the intraportal site the corresponding time was 6 d (range 1–20 d, n ¼ 6). There was no significant difference between the groups (P ¼ 0.4) (Fig. 2A). Assessment of Graft Function

Body weight of animals increased in a similar fashion in the IPtx group and the IMtx group from 277 6 12 g and 274 6 12 g on the day of transplantation, to 427 6 34 g and 420 6 20 g in each group on day 60 after transplantation, respectively (P ¼ 0.8 and P ¼ 0.8, respectively) (Fig. 2B). IVGTT at 8 wk post-transplantation showed a similar temporal blood glucose decline for the IMtx-group, IPtx-group, and controls (Fig. 2C). The glucose elimination rate was impaired in the transplanted animals, resulting in a KG of respectively 1.3% 6 0.2 %/min, 2.2% 6 0.3 %/min, and 3.0% 6 1 %/min for IPtx, IMtx, and controls (controls significantly better than IPtx group, P < 0.05). Similar results were seen in the total 90-minute glucose disappearance rate [KG (1–90)]: 0.6% 6 0.1%/min in the IPtx group, 1.2% 6 0.07%/min in the IMtx group, and 1.5% 6 0.5%/min in controls [controls significantly better than IPtx group (P < 0.01) and IMtx group (P < 0.05)]. Response curves for insulin showed a delayed insulin response in both the IMtx and IPtx group compared with controls (Fig. 2D), but calculated AUCinsulin revealed a similar amount of insulin secreted in the IMtx group and controls [5.0 6 0.5 (U/ mL) 3 h versus 4.2 6 0.7 (U/mL) 3 h, respectively, P ¼ 0.3]. When full function and normoglycemia was obtained it was sustainable to the end of the study at 101 d after transplantation. One animal in the diabetic nontreated control group was sacrificed on day 56 due to fatigue and weight loss. Transplantation of PEGylated Islets

Time to Reversal of Diabetes

Transplantation of syngeneic islets IM resulted in reversal of diabetes in all rats with a median time of 8

Three animals were rendered diabetic and transplanted IM with 3000 PEGylated IEQ as described in the Materials and Methods section. One animal

TABLE 1 Number of Islets (IEQ) that Cured Diabetic Rats Following Intraportal (IP) and Intramuscular (IM) Transplantation

n Cure* Partial function** Non-cure*** *

1000 IEQ IP

2000 IEQ IP

1000 IEQ IM

2000 IEQ IM

3000 IEQ IM

4000 IEQ IM

2 0 0 2

6 6 0 0

2 0 0 2

3 0 0 3

6 0 3 3

6 6 0 0

Cure: nonfasting blood glucose levels persistently <10 mmol/L. Partial function: nonfasting blood glucose levels between 10 and 20 mmol/L. *** Non-cure: blood glucose levels persistently >20 mmol/L. 3 consecutive measurements were required for classification. **

LUND ET AL.: INTRAMUSCULAR ISLET TRANSPLANTATION

149

FIG. 2. Glycemic control after islet transplantation. (A) Reversal of diabetes and maintenance of normoglycemia was measured in chemically diabetic recipients of syngeneic islets into either the intramuscular site (IM, black line, n ¼ 6) (4000 IEQ) or the intraportal site (IP, grey line, n ¼ 6) (2000 IEQ). After islet transplantation, nonfasting blood glucose levels returned to physiological ranges (4.4–7.4 mmol/L) and remained stable long term in both groups, comparable to controls (dotted line, n ¼ 4). Three rats served as diabetic nontreated controls (light grey line, n ¼ 3). (B) Changes of body weight after intramuscular (IM, black line, n ¼ 6) and intraportal (IP, grey line, n ¼ 6) syngeneic islet transplantation and normal healthy control rats (dotted line, n ¼ 2). Three rats served as diabetic non-treated controls (light grey line, n ¼ 3). (C) Blood glucose increments after intravenous glucose load (0.5g/kg body weight) in recipient rats receiving intramuscular islet grafts (IM, black line, n ¼ 4), intraportal islet grafts (IP, grey line, n ¼ 4) on day 56 after transplantation, and normal healthy control rats (dotted line, n ¼ 4). Three rats served as diabetic non-treated controls (light grey line, n ¼ 3). (D) Serum insulin increments after intravenous glucose load (0.5 g/kg body weight) in recipient rats receiving intramuscular islet grafts (IM, black line, n ¼ 4), intraportal islet grafts (IP, grey line, n ¼ 4) on day 56 after transplantation, and normal healthy controls (dotted line, n ¼ 4). Three rats served as diabetic nontreated controls (light grey line, n ¼ 3). Data are presented as mean 6 SD.

obtained partial graft function for 7 d, while the two others did not obtain graft function. Immunohistochemical Analyses

Well-preserved islets with strong insulin immunostaining were observed in all grafts (Fig. 4). In the IMtx-group, single islets (Fig. 4A) or clusters of islets (Fig. 4D) were located between muscle fibers. In the IPtx-group, single islets were located throughout the liver (Fig. 4B). In the IMtx-group, fibrosis developed in relation to clusters of engrafted islets, as evident by acid fuchsin orange G (AFOG) staining (Fig. 4C). Cells from the exocrine pancreas formed small ducts that were relatively abundant in grafts in the IMtxgroup (Fig. 4E and F). To verify graft potency, pancreata from the transplanted animals were harvested

and stained for insulin. Immunohistochemical examination revealed that only a few of the remaining native islets contained insulin positive beta cells (data not shown).

Myocyte mRNA Analyses

The IM transplant procedure volume-dependently upregulated mRNA levels of IL-6 and, to a lesser degree, IL-8 in myocytes harvested 1 hour after initiation of the procedure (Fig. 3A and B). The addition of islet grafts did not aggravate the inflammatory response compared to 0.15 mL transplant medium alone (Tx group, Fig. 3A and B). The transplant procedure did not significantly upregulate mRNA levels of TNFa or IL-1b at this time point (Fig. 3C and D).

150

JOURNAL OF SURGICAL RESEARCH: VOL. 160, NO. 1, MAY 1, 2010

FIG. 3. Effect of the IM transplant procedure on myocyte mRNA levels, measured in muscle biopsies taken 1 hour after initiation of the procedure. IL-6 (A) and IL-8 (B) mRNA levels were volume-dependently upregulated, measured by quantitative real time RT-PCR. The addition of islet grafts (Tx) did not aggravate the inflammatory response compared to 0.15 mL transplant medium alone. IL-1b (C) and TNF-a (D) mRNA levels were not significantly upregulated; 0.15 mL and 0.6 mL: injection of 0.15 mL and 0.60 mL of transplant medium alone, respectively; Tx: injection of 2000 IEQ dissolved in 0.15 mL transplant medium; n ¼ 3 rats per experimental group. The procedure was carried out on the right m. femoris biceps, while the left served as control in each rat. Data are presented as mean 6 SD.

DISCUSSION

There is an increasing emphasis in the islet transplant community on the development of alternative sites for islet implantation that may improve engraftment and provide long-term graft function. In this study, we describe the development of a valid, simple, and reproducible IM islet transplantation model and show that IM islet transplantation to m. biceps femoris is feasible in a syngeneic rat model. Data on the IM site for islet transplantation in the rat are very sparse. We have found only two articles, both almost 30 years old [21, 24]. In those studies a different islet isolation protocol was used as well as different culture conditions and, importantly, the transplantation technique was different; islets were transplanted into pockets in the fascia of the oblique abdominal muscle. Thus, in this context, we interpret our model and findings as new. The development of the model for IM transplantation was challenging until we discovered that very small volumes of islets at different sites in the musculature resulted in partial islet function post-transplant. However, sustained reversal of diabetes was not obtained until a refined pearls-on-a-string implantation technique was developed. With this technique, very small volumes of islets were implanted, aligned at several spots in the muscle.

When utilizing larger transplant volumes (>0.15 mL) or aggregates of islets to one locus in the muscle, a significant post-transplant oedema developed with substantial peri-islet fibrosis as long term result (data not shown). To investigate the mechanism responsible, we tested the immediate effect of the transplant procedure on myocyte gene expression. We found a volume-dependent increase in the expression of IL-6 and, to a lesser degree, IL-8 in myocytes harvested from the transplant area 1 h after initiation of the procedure (Fig. 3A and B). The expression of IL-1b and TNF-a was not significantly upregulated at this time point (Fig. 3C and D). Possible explanations for this finding include: (1) the timing of the biopsy, missing the peak in IL-1b and TNF-a response [31], (2) IL-6 induced increases in IL1 receptor antagonist [32], blunting the IL-1b response, and (3) the magnitude of the TNF-a response to the transplant procedure, as, for example, increases in TNF-a mRNA in skeletal muscle has been reported to be 7- to 10-fold lower than IL-6 increases 2 h after LPS stimulation [33, 34]. IL-6 and IL-8 are pro-inflammatory cytokines/chemokines expressed and produced by myocytes under stress [34–37], and have been implicated in inflammation-induced islet dysfunction [38–43]. It is conceivable that these locally secreted proinflammatory molecules might recruit and activate immune cells to the transplant site, and thereby contribute in causing the

LUND ET AL.: INTRAMUSCULAR ISLET TRANSPLANTATION

151

FIG. 4. Representative sections of muscle and liver from transplanted animals. Islets, stained brown for insulin by immunohistochemistry (ABC-peroxidase), in striated muscle (A) and in liver (B). Bar: 100 mm. Serial section stained for connective tissue (blue) by acid fuchsin orange G (AFOG) (C) and insulin (brown) (D). In this case there is an increased amount of connective fibers (fibrosis) around the islets and ductal tissue. Bar: 200 mm. Cells from exocrine pancreas form small ducts (arrow), stained for fibrosis (E) and insulin (F). Bar: 50 mm.

peri-islet fibrosis observed (Fig. 4C and D). Higher transplant volumes induced larger myocyte proinflammatory response (Fig. 3), underlining the importance of keeping the transplant volumes low. Accordingly, we were able to moderate the fibrosis formation by reducing the transplant volumes and dispersing the islets longitudinally in the muscle. Single islets located

between muscle fibers appeared to have normal morphology (Fig. 4A). Plausibly, post-transplant fibrosis could be further reduced by dividing the islets into yet more portions transplanted to several muscles. In order to try and increase the efficacy of the IM transplant procedure, PEGylation of islets prior to transplantation was evaluated. Initial results did not

152

JOURNAL OF SURGICAL RESEARCH: VOL. 160, NO. 1, MAY 1, 2010

indicate a beneficial effect; however, we are currently evaluating an optimized islet PEGylation protocol, in combination with anti-inflammatory strategies to improve islet survival at the IM site at our laboratory. Examination of IM islet grafts revealed a relative abundance of pancreatic duct cells (PDC) (Fig. 4 E and F). Clinically, the purity of islet preparations transplanted into patients averages 50% to 60% [44], and the total proportion of PDC has been reported to approach 40% [45]. PDC have been suggested to have positive [46], neutral [44], and negative effects [47, 48] on islet transplant outcome. The need for further characterization of PDC in islet transplantation has recently been highlighted [44], and we propose that the IM transplant model may represent a useful tool for evaluating PDC function in islet transplantation. Infusion of foreign cells or organisms into blood triggers injurious reactions, engaging the coagulation and complement systems. We, and others, have demonstrated that probably as much as half of the infused islets are regularly destroyed during the first few minutes after IP transplantation due to such islet-blood interactions [6, 49]. It has also been shown that factors such as islet isolation and ischemia up-regulate inflammatory mediators on islets which contribute and reinforce such islet-blood interactions [6, 7, 43]. Further, when arriving at the liver sinusoids, islets may come in contact with Kupffer cells. Kupffer cells constitute an important part of the reticuloendothelial system (RES), which task includes destruction and phagocytosis of foreign material. In addition, intrahepatic islets are exposed to high portal vein levels of nutrients, gut hormones, and diabetogenic immunosuppressive drugs [9]. As a result, the surrounding hepatocytes will probably be exposed to hypersecretion of undiluted insulin, which may elicit a lipogenic response, resulting in focal steatosis [50]. Intrahepatic islets may therefore be chronically exposed to both a high lipid and glucose environment. Thus, several mechanisms could explain why other implantation sites (e.g., kidney, spleen) show superior long term results compared with IP transplantation [16–19]. We hypothesize that an optimal transplant site should be well vascularised, easy accessible for pretransplant modulation and biopsies, post-transplant excision, and also minimize islet–blood interactions. One of the promising sites proposed is the omental pouch, into which islets can be transplanted laparoscopically [51–54]. However, the procedure involves general anesthesia, risk of post-transplant adhesions, and the site is difficult to access for excision and biopsies if necessary. In addition, there is concern regarding leakage of islets from the pouch. Others have demonstrated reversal of diabetes by islet transplantation into a subcutaneous device, bone marrow, gastric mucosa, and

native pancreas [55–60]. The subcutis is relatively avascular [61], bone marrow and gastric mucosa would probably be difficult to modulate pretransplant, and native pancreas may be difficult to access for biopsies and excision. The kidney and spleen are other promising alternatives, which also have shown superior efficacy compared with intraportal transplantation [17–19]. However, intrasplenic transplantation does not minimize islet–blood interactions, and islets under the renal capsule would be difficult to access for biopsy or excision. In the IM transplantation model, islets are infused into the interstitium between muscle fibers. Hereby, injurious thrombotic reactions due to direct islet-blood contact may be kept at a minimum. The feasibility of the approach has been demonstrated by successful autotransplantation of parathyroid tissue and islet autotransplantation [20, 22]. The experimental IM transplantation model would allow studies on, e.g., bioengineered matrices carrying growth factors that potentially could improve islet engraftment and revascularization. Islets could be tagged and would then be easy to access, and excised if necessary. The time until full graft function tended to be longer in the IMtx group compared with the IPtx group. A similar observation has been made when transplanting islets into an omental pouch [52]. This could be due to differences in the islet engraftment process in musculature compared with that in the liver. A valid and reproducible experimental IM transplantation model could therefore be a tool to study and manipulate the posttransplant revascularisation process. We found the glucose elimination rates following IVGTT to be slightly impaired in transplanted animals (both IMtx- and IPtx groups) compared with controls. The insulin secretory function was impaired, resembling the situation seen after clinical islet transplantation. Alteration in the normal expression of several genes necessary for optimal glucose induced insulin secretion have been proposed following islet transplantation [62], and may be an explanation for these findings. With the current technique for IM islet transplantation, more islets were required to obtain normoglycemia in the IMtx group compared with IP transplantation. We strongly believe that further refinement of the IMtx model will reduce the number of islets necessary for prolonged reversal of diabetes. However, it is known that IP transplantation is more efficient in rats than in humans, presumably due to small-sized liver sinusoids and relatively large rat islets [3]. A more favorable muscle to islet size ratio in humans may imply that IM islet transplantation could work better in humans than in rats. Striated musculature in humans, both in the forearm and, for example m. gluteus, could house large quantities of islets. If an unlimited source of insulin-producing

LUND ET AL.: INTRAMUSCULAR ISLET TRANSPLANTATION

tissue were available, repeated transplantations could easily be performed [63]. In conclusion, the described method of transplanting syngeneic rat islets in a pearls-on-a-string fashion into m. biceps femoris induces reproducible reversal of diabetes and sustained normoglycemia for >100 d posttransplant. The IM islet transplantation model would be accessible for, e.g., various bioengineered matrices carrying growth factors and for composite islet-endothelial cell grafts for promotion of engraftment [23, 64]. The model would also allow application of local immunosuppression and modulators of inflammation. As an experimental model for islet research, we suggest that the IMtx method could be a promising tool for further achievements in the field of islet transplantation. ACKNOWLEDGMENTS This study was supported by the Surgical Department, Transplant Unit at Rikshospitalet University Hospital, Oslo, Helse SØR RHF (3b170), the Norwegian Research Council, Helse og Rehabilitering, Swedish Medical Research Council (16P- 13568 and 16X-12219), the Swedish Diabetes Association, and the Juvenile Diabetes Foundation International. The authors acknowledge the assistance of Helge Scott, Mohammed Shakil Ahmed, Hogne Røed Nilsen, and Martin Brunvand. The authors are grateful to the Nordic Network for Islet Transplantation.

REFERENCES 1. Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med 2006; 355:1318. 2. Ryan EA, Lakey JR, Shapiro AM. Clinical results after islet transplantation. J Investig Med 2001;49:559. 3. Korsgren O, Lundgren T, Felldin M, et al. Optimising islet engraftment is critical for successful clinical islet transplantation. Diabetologia 2008;51:227. 4. Panaro F, Testa G, Bogetti D, et al. Auto-islet transplantation after pancreatectomy. Expert Opini Biol Ther 2003;3:207. 5. Korsgren O, Nilsson B, Berne C, et al. Current status of clinical islet transplantation. Transplantation 2005;79:1289. 6. Barshes NR, Wyllie S, Goss JA. Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: Implications for intrahepatic grafts. J Leukoc Biol 2005;77:587. 7. Moberg L, Johansson H, Lukinius A, et al. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet 2002; 360:2039. 8. Keymeulen B, Gillard P, Mathieu C, et al. Correlation between beta cell mass and glycemic control in type 1 diabetic recipients of islet cell graft. Proc Natl Acad Sci USA 2006;103:17444. 9. Robertson RP. Intrahepatically transplanted islets - strangers in a strange land. J Clin Endocrinol Metab 2002;87:5416. 10. Sohn J, Siegelman E, Osiason A, et al. Unusual patterns of hepatic steatosis caused by the local effect of insulin revealed on chemical shift MR imaging. Am J Roentgenol 2001;176:471. 11. Bhargava R, Senior PA, Ackerman TE, et al. Prevalence of hepatic steatosis after islet transplantation and its relation to graft function. Diabetes 2004;53:1311.

153

12. Dombrowski F, Mathieu C, Evert M. Hepatocellular neoplasms induced by low-number pancreatic islet transplants in autoimmune diabetic BB/Pfd rats. Cancer Res 2006;66:1833. 13. Carlsson PO, Palm F, Mattsson G. Low revascularization of experimentally transplanted human pancreatic islets. J Clin Endocrinol Metab 2002;87:5418. 14. Hogan A, Pileggi A, Ricordi C. Transplantation: current developments and future directions; the future of clinical islet transplantation as a cure for diabetes. Front Biosci 2008;13:1192. 15. Kemp CB, Knight MJ, Scharp DW, et al. Effect of transplantation site on the results of pancreatic islet isografts in diabetic rats. Diabetologia 1973;9:486. 16. Hiller WF, Klempnauer J, Luck R, et al. Progressive deterioration of endocrine function after intraportal but not kidney subcapsular rat islet transplantation. Diabetes 1991;40:134. 17. Mellgren A, Schnell Landstrom AH, Petersson B, et al. The renal subcapsular site offers better growth conditions for transplanted mouse pancreatic islet cells than the liver or spleen. Diabetologia 1986;29:670. 18. Warnock GL, Dabbs KD, Evans MG, et al. Critical mass of islets that function after implantation in a large mammalian. Horm Metab Res Suppl 1990;25:156. 19. Evans MG, Warnock GL, Rajotte RV. Comparison of sites for transplantation of canine pancreatic microfragments. Diabetes Res 1989;10:35. 20. Wells SA, Jr., Gunnells JC, Shelburne JD, et al. Transplantation of the parathyroid glands in man: Clinical indications and results. Surgery 1975;78:34. 21. Weber CJ, Hardy MA, Pi-Sunyer F, et al. Tissue culture preservation and intramuscular transplantation of pancreatic islets. Surgery 1978;84:166. 22. Rafael E, Tibell A, Ryden M, et al. Intramuscular autotransplantation of pancreatic islets in a 7-year-old child: A 2-year followup. Am J Transplant 2008;8:458. 23. Dafoe DC, Wang X, Tafra L, et al. Studies of composite grafts of fetal pancreas (FP) and fetal liver (FL) in the streptozotocininduced diabetic rat. Adv Exp Med Biol 1992;321:171. 24. Axen KV, Pi-Sunyer FX. Long-term reversal of streptozotocininduced diabetes in rats by intramuscular islet implantation. Transplantation 1981;31:439. 25. Liu X, Gu¨nther L, Drognitz O, et al. Persistent normoglycemia in the streptozotocin-diabetic rat by syngenic transplantation of islets isolated from a single donor with Liberase. Pancreas 2006; 32:9. 26. Jahr H, Hussmann B, Eckhardt T, et al. Successful single donor islet allotransplantation in the streptozotocin diabetes rat model. Cell Transplant 2002;11:513. 27. Cadwick DR, Robertson GS, Contractor HH, et al. Storage of pancreatic digest before islet purification. The influence of colloids and the sodium to potassium ratio in University of Wisconsin-based preservation solutions. Transplantation 1994;58:99. 28. Mathews CE, Langley SH, Leiter EH. New mouse model to study islet transplantation in insulin-dependent diabetes mellitus. Transplantation 2002;73:1333. 29. Tiemann K, Panienka R, Klo¨ppel G. Expression of transcription factors and precursor cell markers during regeneration of beta cells in pancreata of rats treated with streptozotocin. Virchows Arch 2007;450:261. 30. Panza JL, Wagner WR, Rilo HL, et al. Treatment of rat pancreatic islets with reactive PEG. Biomaterials 2000;21:1155. 31. Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol 1993;54:1. 32. Steensberg A, Fischer CP, Keller C, et al. IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans. Am J Physiol Endocrinol Metab 2003;285:E433.

154

JOURNAL OF SURGICAL RESEARCH: VOL. 160, NO. 1, MAY 1, 2010

33. Frost RA, Nystrom GJ, Lang CH. Lipopolysaccharide regulates proinflammatory cytokine expression in mouse myoblasts and skeletal muscle. Am J Physiol Regul Integr Comp Physiol 2002;283: R698. 34. Lang CH, Silvis C, Deshpande N, et al. Endotoxin stimulates in vivo expression of inflammatory cytokines tumor necrosis factor alpha, interleukin-1beta, -6, and high-mobility-group protein-1 in skeletal muscle. Shock 2003;19:538. 35. Bartoccioni E, Michaelis D, Hohlfeld R. Constitutive and cytokineinduced production of interleukin-6 by human myoblasts. Immunol Lett 1994;42:135. 36. Hiscock N, Chan MH, Bisucci T, et al. Skeletal myocytes are a source of interleukin-6 mRNA expression and protein release during contraction: Evidence of fiber type specificity. FASEB J 2004;18:992. 37. Lihn AS, Pedersen SB, Lund S, et al. The anti-diabetic AMPK activator AICAR reduces IL-6 and IL-8 in human adipose tissue and skeletal muscle cells. Mol Cell Endocrinol 2008;292:36. 38. Ellingsgaard H, Ehses JA, Hammar EB, et al. Interleukin-6 regulates pancreatic alpha-cell mass expansion. PNAS 2008; 105:13162. 39. Berney T, Molano RD, Cattan P, et al. Endotoxin-mediated delayed islet graft function is associated with increased intra-islet cytokine production and islet cell apoptosis. Transplantation 2001;71:125. 40. Alexandraki K, Piperi C, Kalofoutis C, et al. Inflammatory process in type 2 diabetes: The role of cytokines. Ann NY Acad Sci 2006;1084:89. 41. Campbell IL, Hobbs MV, Dockter J, et al. Islet inflammation and hyperplasia induced by the pancreatic islet-specific overexpression of interleukin-6 in transgenic mice. Am J Pathol 1994; 145:157. 42. Barbe´-Tuana FM, Klein D, Ichii H, et al. CD40–CD40 Ligand interaction activates proinflammatory pathways in pancreatic islets. Diabetes 2006;55:2437. 43. Lund T, Fosby B, Korsgren O, et al. Glucocorticoids reduce proinflammatory cytokines and tissue factor in vitro and improve function of transplanted human islets in vivo. Transpl Int 2008;21:669. 44. Ichii H, Miki A, Yamamoto T, et al. Characterization of pancreatic ductal cells in human islet preparations. Lab Invest 2008 (in press). 45. Keymeulen B, Ling Z, Gorus FK, et al. Implantation of standardized beta-cell grafts in a liver segment of IDDM patients: Graft and recipients characteristics in two cases of insulin-independence under maintenance immunosuppression for prior kidney graft. Diabetologia 1998;41:452. 46. Street CN, Lakey JRT, Shapiro AMJ, et al. Islet graft assessment in the Edmonton Protocol: Implications for predicting long-term clinical outcome. Diabetes 2004;53:3107. 47. Pavlovic D, Chen MC, Bouwens L, et al. Contribution of ductal cells to cytokine responses by human pancreatic islets. Diabetes 1999;48:29.

48. Beuneu C, Vosters O, Movahedi B, et al. Human pancreatic duct cells exert tissue factor-dependent procoagulant activity. Diabetes 2004;53:1407. 49. Eich T, Eriksson O, Sundin A, et al. Positron emission tomography: A real-time tool to quantify early islet engraftment in a preclinical large animal model. Transplantation 2007;84:893. 50. Maffi P, Angeli E, Bertuzzi F, et al. Minimal focal steatosis of liver after islet transplantation in humans: A long-term study. Cell Transplant 2005;14:727. 51. Ao Z, Matayoshi K, Lakey JR, et al. Survival and function of purified islets in the omental pouch site of outbred dogs. Transplantation 1993;56:524. 52. Kenyon N, Berman D, O’Neil J, et al. Long-term survival of cynomolgus monkey islets implanted in an omental pouch site on tissue engineered scaffolds. Am J Transplant 2007;7:572 (abstract). 53. Tuch BE, Sheil AG, Ng AB, et al. Long-term survival of human fetal pancreatic tissue transplanted into an insulin-dependent diabetic patient. Diabet Med 1986;3:24. 54. Yasunami Y, Lacy PE, Finke EH. A new site for islet transplantation - a peritoneal-omental pouch. Transplantation 1983; 36:181. 55. Stagner JI, Rilo HL, White KK. The pancreas as an islet transplantation site. Confirmation in a syngeneic rodent and canine autotransplant model. JOP 2007;8:628. 56. Salazar-Banuelos A, Wright J, Sigalet D, et al. The bone marrow as a potential receptor site for pancreatic islet grafts. Arch Med Res 2008;39:139. 57. Pileggi A, Molano RD, Ricordi C, et al. Reversal of diabetes by pancreatic islet transplantation into a subcutaneous, neovascularized device. Transplantation 2006;81:1318. 58. Halberstadt CR, Williams D, Emerich D, et al. Subcutaneous transplantation of islets into streptozocin-induced diabetic rats. Cell Transplant 2005;14:595. 59. Caiazzo R, Gmyr V, Hubert T, et al. Evaluation of alternative sites for islet transplantation in the minipig: Interest and limits of the gastric submucosa. Transplant Proc 2007;39:2620. 60. Andrades P, Asiedu C, Rodriguez C, et al. Subcutaneous pancreatic islet transplantation using fibrin glue as a carrier. Transplant Proc 2007;39:191. 61. Gu Y, Tabata Y, Kawakami Y, et al. Development of a new method to induce angiogenesis at subcutaneous site of streptozotocin-induced diabetic rats for islet transplantation. Cell Transplant 2001;10:453. 62. Laybutt DR, Hawkins YC, Lock J, et al. Influence of diabetes on the loss of beta cell differentiation after islet transplantation in rats. Diabetologia 2007;50:2117. 63. Hering BJ. Islet xenotransplantation - concepts, challenges, and chances. Xenotransplantation 2007;14:369. 64. Johansson U, Elgue G, Nilsson B, et al. Composite islet-endothelial cell grafts: A novel approach to counteract innate immunity in islet transplantation. Am J Transplant 2005;5:2632.