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THE FATE OF MICROENCAPSULATED RAT ISLETS TRANSPLANTED INTO DIABETIC

MICE

Negda Tabrizi

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology

University of Toronto

@ Copyright by Negda Tabrizi 1998

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THE FATE OF MICROENCAPSULATED RAT ISLETS TRANSPLANTED INTO DIABETIC MICE

Master of Science 1998

Negda Tabrizi

Department of Physiology, University of Toronto

The fate of encapsulated rat islets transplanted into diabetic mice was

investigated. Islets of Langerhans isolated from male Wistar rats were

encapsulated in alginate-polylysine-alginate membranes. One thousar. :

microencapsulated islets were transplanted into the intraperitoneal cavity of STZ-

induced diabetic mice. All mice became normoglycemic at day one

posttransplantation and maintained normoglycemia throughout the study. At 3,

7, and 14 days posttransplantation the encapsulated islets were recovered from

the transplant recipients and analyzed for: insulin content, insulin secretion,

apoptosis, and morphological changes.

Results indicate a significant decrease (pc0.0001) in insulin content and

insulin secretion in the retrieved islets over time. By day 14 posttransplantation

there was no significant difference (p>0.3) in the amount of apoptosis occurring

in the transplanted islets as compared to pre-transplantation values. It is evident

the in vivo environment may be favourable for islets survival. Further research

must be undertaken to completely determine the fate of microencapsulated

islets.

To my dearest parenls, Mohammed Reza and Naoshafarin, and my precious brother Alireza,

for the unconditional love, support and encouragement you have given me through the years

Acknowledqments

I wish to extend my most sincere gratitude to Dr. Anthony M. Sun. Dr.

Sun's continuous guidance and encouragement, invaluable advice and

supervision and persistent leadership and commitment to teaching students have

provided me with a remarkable learning experience. I will always be grateful for

the opportunity I was provided with.

I would also like to thank Dr. Daobiao Zhou for endless hours of patient

teaching, more teaching and even more teaching! 1 will always remember your

kindness.

I also wish to express my gratitude to Dr. Ivan Vacek for guidance,

technical assistance and moral support. I will never forget your ability to alwsys

brighten up my days.

I would also like to thank Teddy, Neggar, Rehan, Winnie, Al, Malathy and

Wendy for moral support, cheerfulness and eagerness to listen. Your

friendships made these ever challenging two years very special.

A final thank you to my supervisory committee Dr. A. Giacca and Dr. C.Y.

Pang and to Dr. W.A. MacKay for priceless advice, comments and suggestions.

TABLE OF CONTENTS

ABSTRACT

ACKNOWLEDGMENTS

TABLE OF CONTENTS

LlST OF ABBREVIATIONS

LlST OF FIGURES

CHAPTER I -Introduction

Diabetes Pancreas lnsulin Chemistry of lnsulin Biosynthesis of lnsulin Insulin Secretion Insulin Action Insulin Metabolism lnsulin Receptor Turnover Type 1 Diabetes Genetics Environmental EffectsNiral Infections Immune Response Current Therapy in IDDM Alternate Strategies for Insulin Replacement in IDDM Pancreas Transplantation Pancreatic Islet Transplantation Protection from Host's Immune System lmmunotherapy lrnmunoisolation and Microencapsulation Fate of Encapsulated Rat Islets Cell Death Necrosis Apoptosis Structural Changes Internal and External Triggers Factors Affecting Apoptosis Fas Mediated Apoptosis Genes Regulating Apoptosis Justification for the Study

ii

iii

iv

vii

viii

i

1 2 3 3 5 6 7 8 9 9 10 10 11 I I 12 12 13 13 13 14 16 17 17 18 18 19 20 21 21 22

CHAPTER 2-Rationale and Hypothesis

2.1 Rationale 2.2 Hypothesis

CHAPTER 3-Methodoloqv and Experimental Desiqn

Methods Diabetic Mouse Model lslet Isolation Encapsulation Transplantation lslet Recovery Insulin Content Determination Glucose Challenge Studies Apoptosis Computer Assisted Morphometry Histology Experimental Design In Vivo Experimental Design Transplantation Studies Diabetic Controls Transplantation of Unencapsulated Rat lslets Transplantation of Empty Capsule Transplantation of Encapsulated Rat lslets Plasma Glucose Measurements Body Weight Determination Capsule Recovery Experimental Groups In Vivo Experimental Groups In Vitro Experimental Groups Analysis Insulin Content lnsulin Secretion Apoptosis Histology Statistics

CHAPTER 4-Results

4.1 Transplantation Studies 4.1 .I Diabetic Control 4. I .2 Transplantation of Unencapsulated Rat Islets 4.1.3 Transplantation of Empty Capsule 4.1.4 Transplantation of Encapsulated Rat Islets 4.2 Insulin Content

Cultured Rat lslets Recovered Rat lslets Comparison of Recovered and Cultured Rat I! Insulin Secretion Cultured Rat lslets Recovered Rat lslets Comparison of Recovered and Cultured Rat I Apoptosis Cultured Rat lslets Recovered Rat lslets

slets

slets

4.4.3 Comparison of Recovered and Cultured Rat Islets 4.5 Electron Microscopy 4.5.1 Recovered Rat Islets 4.5.2 Cultured Rat Islets 4.6 Histology

CHAPTER 5-Discussion

5.1 Transplantation Studies 5.2 Insulin Content 5.3 Insulin Secretion 5 -4 Apoptosis

CHAPTER 6-Conclusion

CHAPTER 7-References

List of Abbreviations

AA ADP APA ATP CAMP C-Peptide ER g GLUT 2 HLA hr I CA IDDM rnM M MW n NlDDM NO RIA ST2 w/v x g

amino acid adenosine di-phosphate aig inate-poly-L-lysine-alginate adenosine tri-phosphate cyclic-Adenosine monophosp hate connecting peptide endoplasrnic reticulum gram Glucose Transporter 2 Human Leukocyte Antigen hour islet cell antibodies insulin dependent diabetes mellitus miIlimoIar molar molecular weight number of subjects or replicates non-insulin dependent diabetes rnellitus nitrogen oxide radioirnmunoassay streptozotocin weight per volume centrifugal force

vii

List of Figures

Figure I:

Figure 2:

Figure 3:

Figure 4:

Figure 5:

Figure 6:

Figure 7:

Figure 8:

Figure 9:

Experimental Design Flow Chart

lnsulin Content Flow Chart

lnsulin Secretion Flow Chart

Apoptosis Flow Chart

Blood Glucose Profiles of Diabetic Control Mice

Blood Glucose Profiles of Diabetic Mice Transplanted with Unencapsulated Rat Islets

Blood Glucose Profiles of Diabetic Mice Transplanted with Empty Capsules

Blood Glucose Profiles of Diabetic Mice Transplanted with Encapsulated Rat lslets for 3 Days

Blood Glucose Profiles of Diabetic Mice Transplanted with Encapsulated Rat lslets for 7 Days

Figure 10: Blood Glucose Profiles of Diabetic Mice Transplanted with Encapsulated Rat lslets for 14 Days

Figure I 1 : Body Weight Determination of Diabetic Control, Empty Capsule and Encapsulated Rat Islet Transplanted for 14 Days

Figure 12: Photomicrograph of Encapsulated Rat lslets Recovered at 14 Days Posttransplantation (LM 130X)

Figure l3a: lnsulin Content, ln Vitro Cultured lslets

Figure 13b: lnsulin Content, Recovered lslets as Compared to Untransplanted Controls

Figure 13c: lnsulin Content: In Vitro Versus Recovered Rat lslets

Figure 14a: Glucose Challenge, In Vitro Cultured lslets

- -. Vl l l

Figure 14b: Glucose Challenge, Recovered Islets as Compared to 77 Untransplanted Controls

Figure Ma: Percentage of Apoptotic Nuclei in in Vitro Cultured lslets 81

Figure 15b: Percentage of Apoptotic Nuclei in Recovered Islets as 83 Compared to Untransplanted lslets

Figure 15c: Apoptosis: In Vitro Versus Recovered Rat Islets 85

Figure 16a: Apoptotic P cell of the recovered rat islets at 7 88 days posttransplantation.

Figure 16b: Normal P cells of recovered rat islets at 7 days posttransplantation.

Figure 16c: Necrotic p cell of recovered rat islets at 7 days posttransplantation.

Figure 17a: Apoptotic and normal viable P cells in 7 day in vitro cultured rat islets.

Figure 17b: Necrotic P cell of 7 days in vitro cultured rat islets. 96

Figure 18a: Encapsulated rat islets I day in culture stained with 99 aldehyde fuchin and neutral red.

Figure 18b: Encapsulated rat islets recovered at 14 days I01 posttransplantation stained with aldehyde fuchin and neutral red.

Figure 18c: Encapsulated rat islets in vitro cultured for 14 days 103 stained with aldehyde fuchin and neutral red.

1. INTRODUCTION

1 .I Diabetes

Diabetes mellitus represents a chronic disorder of metabolism and is

characterized by absolute or relative lack of insulin production by the pancreas.

Diabetes is identified by hyperglycemia, polyuria, polydipsia, weight loss.

glucosuria, ketosis, acidosis and coma. The long term complications associated

with diabetes include retinopathy, nephropathy, neuropathy, cardiac problems,

hypertension, infections, impotence and pregnancy complications. Diabetes

mellitus has two forms: type 1 {formerly known as insulin dependent diabetes

rnellitus (IDDM)} and type 2 {formerly known as non-insulin dependent diabetes

mellitus (NIDDM)}. Type 1 and 2 diabetes differ in the mechanism of insulin

deficiency and age of onset. Type 1 diabetes generally begins at an early age

(juvenile onset) and is insulin dependent whereas type 2 diabetes begins later in

life (maturity onset) and is often insulin independent. Gestational diabetes is a

temporary condition that occurs during pregnancy and women with gestational

diabetes have a greater chance of developing type 2 diabetes later in life.

Before introducing the subject of this study, the pancreas as well as the role of

insulin and complications caused by a lack of insulin in type 1 must first be

examined.

1.2 Pancreas

The pancreas consists of two different parts, the exocrine and the

endocrine. The exocrine system is primarily made of acinar tissue that secretes

enzymes and ions into the duodenum to be used for digestive purposes. The

endocrine portion of the pancreas secretes four hormones and carefully

regulates blood sugar levels. The islets of Langerhans make up the endocrine

portion of the pancreas. lslets are scattered throughout the pancreas although

they are mostly concentrated in the tail rather than the head or body of the

pancreas (1-4). lslets compose 14% of the total weight of the pancreas and, in

humans, there are about 1-2 million islets. Each islet has a copious blood supply

and blood from the islets is released into the pancreatic veins that drain into the

hepatic portal vein. lslets consist of four different cell types, a, P, 6 and F cells.

The four cell types constitute approximately 3000 cells per islet. lslets range in

size from 100 to 300pm (1). a cells produce glucagon, a catabolic hormone

mobilizing glucose into the bloodstream. P cells produce insulin, an anabolic

hormone, which increases the storage of glucose, fatty acids and amino acids. 6

cells produce somatostatin, a hormone that plays a role in the regulation of islet

cell secretion and F cells secrete pancreatic polypeptide that may have a role in

gastrointestinal function. P cells account for 60-80% of the cells in the islet and

are centrally located. a cells surround the P cells and comprise 15-20% of the

cells in the islet, while 6 and F cells are scattered throughout the islets making up

less than 5% of the cells.

1.3 Insulin

Insulin is a peptide hormone produced by the P cells of the islets of

Langerhans. Insulin, along with the counterregulatory hormones, primarily

glucagon, is actively involved in regulating blood glucose and metabolite levels.

Insulin acts to lower blood glucose levels by specifically targeting hepatic,

adipose and muscle tissue. The failure to secrete insulin in type 1 diabetes has

profound effects on the diabetic subject who consequently requires exogenous

insulin to help in regulating blood glucose levels and restoring energy to the cells.

1.3.1 Chemistry of Insulin

The insulin molecule contains two polypeptide chains. The A chain

contains 21 amino acids while the B chain contains 30 amino acids. The two are

linked by two disulfide bridges that connect A7 to B7 and A20 to B19 (1,3-5). In

addition, there is a third disulfide bridge that connects two residues on the A

chain, in particular residues 6 and 11 (1,3-5). The complete molecule contains

51 amino acids and has a molecular weight of approximately 6,000 daltors.

There are minor interspecies differences in the amino acid composition of

insulin, but the biologically active region of the molecule (the location of the 3

disulfide bridges, the hydrophobic residues in the C-terminal region of the B

chain and N- and C-terminal region of the A chain) is invariant. Porcine insulin

differs f r ~ m human insulin by only one amino acid, a terminal alanine instead of

threonine in the B chain. Bovine insulin differs from human insulin by three

amino acids, alanine replaces threonine at position 8, valine replaces isoleucine

at position 10 in A chain and there is a terminal alanine instead of threonine in

the B chain. The treatment of type 1 diabetes with insulin from a heterologous

species causes an immune response in which anti-insulin antibodies from the

recipient inhibit the activity of the administered insulin. However, this problem

has been overcome with the use of recombinant technology to produce human

insulin and replace pig and bovine insulin as exogenous insulin therapies for

humans.

Although A and 6 chains of insulin have been conserved throughout

species, C peptide has not. Even though the human and porcine insulin

molecule differ only by 1 amino acid, the C peptide of the porcine insu:in

contains 10 more residues and 2 more amino acids (3). Once the function of the

C peptide is determined, perhaps it can be speculated why the C peptide has not

been conserved across species.

Insulin is usually a monomeric protein. Factors such as pH, osmolality

and zinc concentration can cause insulin to self associate (1). When conditions

favour self-association, dimers form via hydrogen bonds between 8-24 and B-

26 on adjacent insulin molecules. Three dimers can also aggregate side by side

to form a hexamer. In mammalian insulin the presence of zinc accelerates

hexamer formation. Usually two zinc molecules along with their attached 12

insulin molecules aggregate (1). Formations of two zinc insulin hexamers are

important for insulin storage, especially in secretory vesicles inside P cells.

When examined under electron microscopy, secretory granules contain an

electron dense core correlating to the position of the insulin and zinc, surrounded

by a pale, clear halo region that contains the C peptide.

1.3.2 Biosynthesis of Insulin

Insulin is transcribed and translated as a preprohormone (M.W. 11,500)

from the insulin gene. The gene is located on the short arm of chromosome 11

in humans and has 3 exons and 2 introns (4). Most mammals express an insulin

gene similar to the human gene, however rats and mice have 2 nonallelic genes

each coding for a unique proinsulin that is processed into two distinct active

insulin molecutes.

The human preproinsulin has a 23 amino acid signal peptide that is

cleaved by an endopeptidase, signal peptidase, that is localized on the cisternae

of the rough endoplasmic reticulum (RER) of the P cells (1). In the RER, the

proinsulin polypeptide (M.W. 9,000) forms disulfide bridges and folds with the

help of C peptide. The proinsulin molecule is transported to the Golgi apparatus

where proteolysis brea~s proinsulin into equimolar amounts of insulin and C

peptide. Both peptides are then packaged into secretory vesicles (6.7).

Granules continue to mature as they transverse the cytoplasm towards the

plasma membrane. Glucose stimulated insulin secretion signals the granules to

fuse with the plasma membrane and release their contents into the extracellular

fluid by a specialized form of exocytosis known as emicytosis. Insulin then

crosses the basal laminae of the P cell and a neighboring capillary, entering the

bloodstream through the fenestrated endothelium of the capillary.

1.3.3 Insulin Secretion

lnsulin secretion can be affected by glucose, amino acids, fatty acids,

ketone bodies, hormonal factors, and pharmacological agents (3.5). An increase

in glucose concentration is the most important physiological regulator of insulin.

Insulin secretion is biphasic. There is a fast or first phase response (5-10

minutes) begins within one minute of sensing glucose. Following the first phase

there is a more gradual prolonged response that lasts until the glucose stimulus

is removed. High protein and fat meals also stimulate insulin secretion. Some

amino acids such as, arginine, lysine and leucine are potent insulin

secretagogues whereas fatty acids and ketone bodies are weak secretagogues.

Many hormonal factors affect insulin secretion. Epinephrine and somatostatin

in hibit insulin release whereas glucagon, GIP, GLP-1 and P adrenergic agonists,

such as isoproterenol, stimulate insulin secretion via adenylate cyclase-CAMP

pathway (1). Prolonged exposure to growth hormone, cortisol, placental

lactogen, estrogen and progestins also stimulate insulin secretion. Sulfonylurea

compounds and other pharmacological agents also act to increase insulin

secretion (3,5).

The mechanism for insulin secretion begins with the entry of glucose into

the p cell via facilitated diffusion through a low affinity (high Krn) glucose

transporter, GLUT 2, (4,8). Once inside the P cell, glucokinase (high Km)

converts glucose to glucose-6-phosphate (G-6-P) (the rate-limiting step). G-6-P

enters the glycolysis pathway resulting in an increase in the intracellular

ATP:ADP ratio (1). ATP-sensitive Kc-channels, that are usually open to allow K+

to exit the cell, close due to increased ATP levels. This closure causes an

increase in intracellular K+ levels which depolarizes the cell membrane-

Depolarization activates voltage sensitive ca2+-channels, opening them and

allowing ca2' to rush into the P cell (1). The calcium is required for the

exocytosis of the insulin-containing secretory vesicles although the exact

mechanism is not completely understood.

1 -3.4 Insulin Action

Insulin enters the circulation and affects tissues expressing the insulin

receptor. The insulin receptor is a tyrosine kinase heterodimer containing two

regulatory extracellular a (M.W. 135,000) and two catalytic membrane spanning

P (M.W. 95,000) subunits (9). The insulin receptor subunits are synthesized

from a single mRNA from chromosome 19 that contains 22 exons. The

synthesized polypeptide is proteolytically separated and rearranged to form the

receptor unit via disulfide bonds (4). lnsulin binds to the a subunit extracellularly

and triggers the autophosphorylation of the p subunit. Primarily, the

phosphorylated insulin receptor phosphorylates insulin receptor substrate4

(IS-1). IRS-1 is a cytoplasmic protein (M.W. 131,000) which is the specific

substrate for insulin, IGF-1, and 114 receptors. IRS-1 is widely expressed in

tissues, highly conserved among species and contains multiple tyrosine

phosphorylation sites (9). IRS-1 acts as a docking protein for several

intracellular enzymes and adapter molecules and it is the docking of IRS-1 to

these proteins that initiates the insulin action cascade. The cascade results in

translocation of glucose transporters (GLUT 4 in muscle and adipose) (4,A 0-14);

stimulation of protein synthesis; inhibition of protein degradation; lipid storage

(inhibition of lipid meta~olism); activation of glycogen synthesis and glycoly;ic

enzymes (liver and muscle); growth and gene expression (via Raf and MAPKK)

(I 2, 15-1 7).

lnsulin action involves the three metabolic fuels for energy, carbohydrate,

protein and fat and occurs in three principal tissues, liver, muscle and adipose

tissue. Insulin increases glucose entry in adipocytes and muscle by translocating

GLUT 4 to the plasma membrane from storage vesicles (10). A pool of GLUT 4

is maintained in the cytoplasm of adipocytes and muscle fibers and when these

cells are exposed to insulin, the transporters rapidly move to the cell membrane

by exocytosis. When the stimulation stops, they are recycled back to the

cytoplasm via endocytosis (4,9,'ll, I3,A4,18). In adipose tissue there is an

increased triglyceride deposition (activation of lipoprotein lipase (LPL) and

inhibition of hormone-sensitive lipase (HSL)) (4,19,20). In muscle there is an

increase in glycogen synthesis, amino acid uptake and protein synthesis. In

general insulin promotes anabolic activity.

1.3.5 Insulin Metabolism

Insulin has no plasma carrier protein and thus has a very short plasma

half-life, less than 3-5 minutes in humans (3-5,9). The mechanism of insulin

metabolism involves a wo-enzyme system and occurs primarily (-80%) in the

liver, kidneys and placenta (21,22). The first enzyme involved is an insulin

specific protease. Although the protease is found in many tissues, it is highly

concentrated in the above mentioned organs. The second is hepatic

glutathione-insulin transnydrogenase which reduces the disulfide bridges leaving

the a and p subunits to degrade rapidly.

1.3.6 Insulin Receptor Turnover

Following the biding of insulin to its receptor, the hormone-receptor

complex enters the cell via receptor-mediated endocytosis. The

hormonelreceptor complexes enter lysosomes (3) where some receptors are

broken down (4). However, most of the receptors are not degraded but rather

recycled (3). The normal turnover time for the insulin receptor is 7 to 12 hours

however in the presence of insulin, the turnover dramatically decreases to 2 to 3

hours (3).

I .4 Type I Diabetes

Type 1 diabetes is caused by an autoimmune disease with anti-p cell

antibodies leading to the destruction of the P cells of the islets of Langerhans

which results in the deficiency and eventual loss of insulin secretion.

There is a specific sequence of events involved in type 1 diabetes

beginning with a genetic predisposition to developing type 1 diabetes (3).

However, a genetic predisposition alone does not cause diabetes. Acquired

environmental factors or viral infections trigger an autoimmune response

beginning with insulitis. The final stage in acquiring diabetes is P cell injury and

death.

1 -4. I Genetics

The major histocompatibility (HLA) complex in humans consists of four

gene loci: A, B, C, and 3. Variation in genes that are located within or near the

HLA complex (3,23,24,, on the short arm of chromosome 6, are a major

component in the susceptibility to type 1 diabetes. Type 1 diabetes studies

initially showed an association with alleles of HLA class B loci. Studies have now

shown that type 1 diaberes individuals are genetically predisposed to express the

histocompatibility alleles in section DR (D related locus), in particular DR3, DE4

or both. The relative risk for developing type 1 diabetes is greater for individuals

who posses both DR3 and DR4 (heterozygotes) than for individuals who are

DR3/3 or DR414 (homozygotes) (3,23,24). Interestingly, there is no association

between specific HLA types and type 2 diabetes.

1.4.2 Environments I EffectsNiral Infections

It has been suggested that certain viral infections (3,25-27) or

environmental conditions may trigger the expression of the HLA-DR alleles on

the pancreatic p cells which may allow the association of an autoantigen to a T

cell. This association is followed by activation of autoreactive immunocytes

(3,261.

1.4.3 Immune Response

lnsulitis is also known as lymphocyte infiltration of the pancreas. The

lymphocytes target and infiltrate the pancreatic islets that still contain P cells

After insulitis, the auto!mmune response is detected with circulating islet cell

antibodies (ICA) directed against P cell surface antigens. This process proceeds

until most, if not all, islers have been destroyed. Recent evidence suggests that

Fas, an apoptosis inaucing surface receptor involved in controlling tissue

homeostasis and function, is expressed extensively in patients newly diagnosed

with type 1 diabetes. This observation suggests indicates that the death

mechanism involved in type 1 diabetes is apoptosis (28-30).

1.5 Current Therapy in Tv~e 1 Diabetes

An effective treatment in type 1 diabetes is administration of insulin. As

insulin is broken down by the proteolytic enzymes in the gastrointestinal tract, it

is not effective if taken orally and therefore must be injected subcutaneously (2).

Insulin absorption can be affected by the injection site, exercise, the accuracy of

the dosage measurement, depth of injection and by environmental temperatures.

There is rapid action, intermediate action and prolonged action insulin. Rapid

action insulin consists of regular and semilente insulin with peak activities at 1-3

hours and 3-4 hours respectively. Lente and NPH, intermediate action insulin,

peak at 6-14 hours and prolonged action ultralente insulin peaks at 18-24 hour.

Proper management of type 1 diabetics with insulin therapy requires patient

familiarity with the types of insulin preparations available and recognition of the

need of individualization of the treatment to meet the metabolic, psychological

and social needs of the patient. Insulin therapy programs can range from one or

two up to three or four injections per day with any insulin combination. In fact.

studies from the Diabetes Control and Complications Trial (DCCT) revealed that

four daily insulin injections significantly reduced blood sugar levels and rates of

kidney, eye and nerve carnage (31) though patients were more likely to develop

hypoglycemia. In general, insulin injections are given a half hour before meals to

help combat the increased blood glucose levels after meals.

1 -6 Alternative Strategies for insulin Replacement Therapy

1.6.1 Pancreas Transplantation

Patients in the late stages of diabetes with end-stage renal failure require

kidney transplants. It is generally under these circumstances that simultaneous

pancreas transplants are performed. Pancreas, either whole or segments, are

grafted with the portal vein. The elimination of exogenous insulin and the return

to normal insulin secretory responses to meal ingestion are indicative of a

functioning P cell mass. The problems associated with pancreas transplantation

and whole organ transplants include thrombosis, ascites, infection, abscess and

hemorrhage (3) and above all, the need for immunosuppression. In addition,

poor organ availability is also a serious concern.

Pancreatic Islet Transplantation

Pancreatic islets transplantation has become an alternative to whcle

pancreas transplants, as it does not require extensive surgery and the bulk of the

pancreas, the exocrine portion, has been removed. In addition, islet

transplantation is less costly than whole or partial pancreas transplants. With the

advent of pancreatic islet isolation (32), islets from rats, mice, pigs and humans

have been isolated and both allo- and xenotransplantations have been

performed. Since 1990, approximately 145 islet allotransplantations have been

performed on human patients (33). Various transplantation sites have been

tried, including injections into the portal vein, intraperitoneal injections and

transplantation under the kidney capsule. While allotransplantations are more

effective than xenotranspiants, the majority of transplanted islets in both cases

have lost function due to immunorejection. A feasible solution to this problem is

to isolate the pancreatic islets in a semipermeable membrane that provides a

physical barrier against i h e host's immune system.

I .7 Protection from Host's Immune Svstem

1.7.1 lmmonotherapy

lmmunosuppressive agents may prevent, attenuate, or retard the onset of

type 1 diabetes or induce remission in a patient who already has the disease.

lmrnunosuppression with drugs, such as cyclosporin, produces amelioration of

type 1 diabetes if given early enough in the course of the disease before all the P

cells are lost (3). Cyciosporin has a number of undesirable side effects

especially nephrotoxicity. Although cyclosporin hinders the progression of type 1

diabetes, the impairment of renal function indicated by increased serum

creatinine levels causes much concern. The disadvantage of

immunosuppressive drugs is that the patient's immune system becomes

significantly compromised, thereby making the individual vulnerable to infection.

1.7.2 lmmunoisolation and Microencapsulation

Chang was the first to propose the concept of artificial cells that would

function to replace an organ (34). Encapsulation of transplanted cells obviates

the need for immunosuppressive drugs because the cells are immunoisolated

from the recipient. The transplanted cells are enclosed in a semipermeable

membrane that allows nutrients, gases and hormones to permeate the capsule

while simultaneously obstructing the entry of larger molecules, such as

immunoglobulins. Sun and Lim (35) reported, for the first time, that an

intraperitoneal transplantation of microencapsulated islets of Langerhans

corrected hyperglycemia in diabetic animals and thus introduced the concept of

islet microencapsulation as a treatment for diabetes. Since its introduction,

numerous studies have been reported by Sun (35-48), Soon-Shiong (48,49),

Calafiore (50) and others supporting the use of microencapsulated islets as a

vehicle for reversing the diabetic conditions in animals.

The use of the original air-jet technique in capsule formation resulted in

capsules that were 0.8-1.0mm in diameter. An improvement to the older air-jet

technique, the eiectrostatic droplet generator, was developed by Sun (51). The

electrostatic droplet generator produces capsules with smaller diameters, 0.25-

0.35rnrn. The advantages of the smaller capsule include increased cell viability,

because cells have improved access to oxygen and nutrients; faster response to

glucose fluctuations, because of decreased dead space; a reduction in volume of

capsules needed for transplantation and less susceptibility to overgrowth on the

surface of the capsule. In addition, the capsules have improved sphericity and a

smoother surface.

For the microcapsule to be effective in the immunoisolation of

transplanted tissue, it has to be biocompatible. The hydrogel nature (85% w/v

H20) of the capsule constructed from alginate-poly-L-lysine-alginate contributes

to its biocompatibility (45,52,53). Furthermore, the reduction of frictional irritation

to the surrounding tissues by the soft pliable consistency of the capsule also

contributes to its biocompatibility. In addition, alginate-poly-L-lysine-alginate

microcapsules have been shown to have a smooth surface (45). The

smoothness of the outer surface inhibits cell attachment, enabling the

microcapsules to remain semipermeable and effective inside the body for long

periods of time.

In order for the capsule to be successful in immunoisolating the

transplanted cells from the host's immune system, the size of the pores in the

membrane need to be controlled. Pore size should be large enough to allow for

the diffusion of nutrients, hormones and gases (glucose, 02, COz), but small

enough to prevent the passage of immunoglobulins (45.53). The permeability of

the membrane is predominantly dependent upon the molecular weight of poly-L-

lysine (PLL) utilized (45,5334). The greater the molecular weight of PLL. t k

greater the pore size. rlowever, if the molecular weight of PLL is too low, the

membrane strength is compromised, due to fewer cross linkages being formed

between the shorter PLL. chains (54).

For the long term survival of encapsulated cells, the strength of the

membrane is important. The strength of the capsule is correlated to size (53); a

smaller capsule is stronger than a larger capsule. Additionally, the smaller

capsule has the advantage of causing less irritation to the surrounding tissue.

Smaller capsules have been shown to significantly increase the duration of

normoglycemia (43).

The level of invasiveness involved in the transplantation of

microencapsulated islets is minimal (intraperitoneal injection), thus decreasing

the trauma experienced by the recipient. Once inside the peritoneal cavity, the

semipermeable capsule allows the cells to respond to physiological conditions

without being detected 2nd destroyed by the recipient's immune system.

1.8 Fate of Microencapsulated Rat Islets

In earlier studies (35-47) it was shown that if a sufficient number of

encapsulated islets art3 transplanted in diabetic animals, hyperglycemia is

reversed and normoglycemia achieved. In addition to secreting insulin.

pancreatic islets release glucagon, somatostatin and pancreatic polypeptides in

response to physiological demands. The use of microencapsulated islets as a

vehicle for treating diabetes in experimental animals has proven to be very

effective. However, the changes that occur in the encapsulated islets in the

period following transplantation remain unclear. To date, very little information

about such changes is available. Islets normally have a copious blood supply

and when isolated by collagenase digestion, their normal environment is

drastically changed. Once encapsulated, islets rely on the diffusion of oxygen

and nutrients for their survival. Eventually, islet function does deteriorate and

islets begin to die, returning hyperglycernia. It is very likely that P cells in the

centre of the islets will be subjected to some degree of hypoxia. Hypoxia is

known to have detrimental effects on cell survival, causing cell necrosis. There

has been much controversy recently as to whether islets die by necrosis or

apoptosis in the period following transplantation (92, 100). The purpose of this

study is to determine the changes that occur in the encapsulated islets in the

period following transplantation and to determine whether apoptosis is the cause

of cell death in the islets. In addition, the changes occurring in the transplanted

islets will be compared with encapsulated islets maintained in culture. There are

major distinguishing signs that differentiate apoptosis (programmed cell death)

from necrosis (accidental destruction).

1.9 Cell Death

1.9.1 Necrosis

Necrosis occurs when the cell has been severely injured (55). Such injury

can be due to physical damage, oxygen deprivation, toxicity through pH, extreme

temperatures or invasion by foreign agents. In such situations, because the cell

cannot control fluid and ion balance, the imbalance causes extreme swelling

both within the cell and the intracellular organelles (especially the

mitochondrion). Necrosis is often described as "the cell balloons and then

ruptures." The extreme swelling of the cell degrades the membrane and the

organelles (56). This degradation leads to an inflammatory response (55,56).

Circulating macrophages and white blood cells converge on the necrotic cells

and ingest them. The ingestion helps limit infection and clear away any leftover

debris. Although white blood cells help in removing the disrupted cells, their

actions and secretions can also lead to excessive damage of the surrounding

normal tissue (55).

1.9.2 Apoptosis

Cell number is aetermined by a balance between cell proliferation and

elimination. Apoptosis is one mechanism for cell elimination. Apoptotic cell

death plays a crucial role in embryogenesis, normal tissue turnover, immune

development and defense and protection against tumorigenesis. The cell death

pathway is activated when the cell is no longer needed or has sustained serious

damage.

1 -9.2. I Structural Changes

Apoptosis, programmed cell death, is quite different from necrosis. For

apoptosis to occur, three sequential processes must occur. First, the cell must

receive the signal to die. Second, the cell must activate its endogenous death

machinery and lastly the cell must be removed following death.

In addition to the sequential processes in apoptosis, major differences

exist between apoptosis and necrosis. There is no swelling in apoptosis.

Apoptotic cells shrink and pull away from neighbouring cells or macrophages

(55.57). The dying cells begin to bleb and are ingested by neighbouring

scavenger cells (55,58). In the nucleus, the chromatin condenses and forms a

bleb near the nuclear envelope (56-64). Apoptotic cell death is an active death

due to cellular energy expenditure to complete the process (55). Apoptosis is

thought to activate or begin de novo synthesis of endonuclease(s), which

specifically cleave DNA at linker regions between nucleosomes to form

fragments, called apoptotic bodies (56-58,60,65-69). The apoptotic bodies are

approximately 180-200 oase pairs of double stranded DNA (60). This cleavage

is thought to be the key biochemical event in apoptosis and is used in its

detection (70). Because of the extensive RNA and protein synthesis for the

endonuclease, it is believed that death genes are turned "on" and survival genes

"offt (58,71). Another distinguishing characteristic of apoptosis is that there is no

inflammation (55,58,62) or sign of lymphocyte infiltration (59).

1.9.2.2 internal and External Triggers

The trigger for apoptosis can be an internal or external stimulus. The

withdrawal of trophic factors and change in the hormonal environment of

hormone dependant tissues (57.72) as well as cell surface interactions with

specific apoptosis receptors, exemplify some external stimuli. Internal stimuli

include exposure to toxins and metabolic or cell cycle disruptions (73). The

apoptotic process is a rapid one and morphological evidence is very short lived.

It is therefore very critical to make observation at the right time in order to be

able to detect apoptosis.

I .9.2.3 Factors Affecting Apoptosis

The treatment of hormone-dependant tissue with calcium channel

blockers or TGFP induces apoptosis (72). Interleukin 1 (IL-I ), tumour necrosis

factor-alpha (TNF-a) and interferon-gamma (IFN-y) may cause P cell dysfunction

that lead to DNA damages destroying the insulin producing cells (74-76). The

cytotoxic effects of cytokines are associated with the exaggerated generation of

nitric oxide (NO) which has been proposed to induce apoptosis whether it is

endogenously produced or in response to cytokines (77). IFN-y's apoptotic

effects are not med iaied through NO (76). N-nitro-L-arginine methylester

(NAME), nicotinamide (NA) or a combination of NAME and NA have been shown

to inhibit the production of NO by competitively inhibiting nitric oxide synthase

(NOS) (78.79) and preventing IL-1 induced inhibition of insulin secretion (75,80).

NAME and NA have been shown to reduce the percentage of cells exhibiting

DNA strand breaks indicating that NO has direct involvement in DNA

fragmentation associated with apoptosis. Interestingly, insulin-like growth factor

'1 (IGF-1) decreases cytokine induction of NOS thereby protecting islets against

cytokine-med iated apoptotic cell death (8 1).

1.9.2.4 Fas Mediated Apoptosis

NO is thought to upregulate the Fas receptor, an apoptosis-inducing

surface receptor (28). Fas receptor belongs to the tumor necrosis factor receptor

family (82,83). Fas ligand expressed on the cytotoxic T lymphocyte surface, is

activated through the binding of its receptor on cell surfaces. Upon binding, the

cytotoxic T lymphocyte releases the contents of its granules, mainly perforin and

serine protease granzyme B (34). Perforin punches holes in the target cell

allowing granzyme B to enter. Granzyme B begins the signalling pathway that

converges with the activation of proteolytic enzymes known as caspases or ICE-

like proteases (interleukin 1 converting enzyme) (83,84). The proteases cleave

intracellular proteins both in the nucleus and cytoplasm and therefore digest the

cell from within.

f -9.2.5 Genes Regulating Apoptosis

In apoptosis there is increased expression of transforming growth factor 1

(TGFPI) gene and protein, thus TGFPl has been considered a good marker of

apoptosis (65,72,85). TGFP I protein can induce apoptosis in hepatocytes

(86.87) and inhibit epithelial cell replication. In addition, the p53 gene normally

functions to protect the host cell through the induction of apoptosis if the cell has

received genotoxic damage. Many carcinomas cause a mutation in the p53

gene in hibiting apoptosis (88). Unlike the TGFP I protein, which increases

apoptosis, bcl-2 and bcl-xL oncoproteins suppress hypoxia-induced apoptosis

(89) through an unknown mechanism, not through the regulation of reactive

oxygen species (ROS) (63,71).

1.10 Justification for the Study

As the beginning of human clinical trials are quickly approaching, a more

complete understanding of the fate of encapsulated islets is necessary. By

examining the amount of cell death that is due to apoptosis and/or necrosis.

encapsulated islet viability in the immediate posttransplantation period can be

assessed. This assessment will better prepare researchers for the long term

studies of the fate of the encapsulated islets. Once a complete understanding of

the fate of the transplanted encapsulated islets is obtained, the number and

frequency of transplantations human patients will require to reverse diabetes will

be easier to evaluate.

2.1 RATIONALE

The changes that occur in the encapsulated islets in the period following

transplantation remain unclear. To date, very little information about such

changes is available. There has been much controversy recently as to whether

islets die by necrosis or apoptosis in the period following transplantation (92,

100). The purpose of this study is to determine the changes that occur in the

encapsulated islets in the period following transplantation and to determine

whether apoptosis is the cause of cell death in the islets. In addition, the

changes occurring in the transplanted islets will be compared with encapsulated

islets maintained in culture.

2.2 HYPOTHESIS

It was hypothesized that the degree of islet cell death in encapsulated rat

islets will be kept at such a level whereby the overall physiological competence

of the transplanted islets will not be significantly compromised (i-e. apoptosis will

not significantly increase). It was also hypothesized that the encapsulated islets

are more functional in in vivo conditions as opposed to in vitro culture.

CHAPTER 3

3. METHODOLOGY & EXPERIMENTAL DESIGN

3.1 Methods

3.1 .I Diabetic Mouse Model

Male C57BU6 mice (Charles River, St. Constant, PQ) weighing 15-209

were allowed to acclimatize for one week before the induction of diabetes.

Diabetes was induced using streptozotocin, which targets and destroys the P cell

of the pancreas (go), :bus producing a chemically induced model of type 1

diabetes. Streptozotocm was dissolved in 0.1M sodium citrate buffer (pH 4.5)

and administered by a single tail vein injection (1 85mglkg body weight). Blood

glucose measurements were taken daily and only those animals registering three

consecutive non-fasting blood glucose measurements above 2OmmollL were

used as recipients for the study.

3.1.2 Islet Isolation

Islets of Langerhans were isolated from male Wistar rats (Charles River,

St. Constant, PQ) weighing 325-3759 using a modified version of the technique

of Lacy and Kostianovsky (32). Briefly, under Ketamine HCI (120mg/kg, MTC

Pharmaceuticals, Cambridge, ON) and Xylazine (25mg/kg, Chemagro Ltd.,

Etobicoke, ON) anesthesia a midline laparotomy from the xiphoid process to

pubis was made to expose the peritoneal cavity. In order to better expose the

cavity, the ends of the midline incision were extended laterally. The common bile

duct was located and clamped at its opening closest to the duodenum and the

rats were then exsanguinated by cutting the abdominal aorta and vena cava.

The beveled tip of a PE-20 catheter, attached to a lOmL syringe containing

3.5mg of Collagenase XI (Sigma Chem Co., St. Louis, MO) in IOmL of MHBSS

was inserted into the common bile duct at its end closest to the liver and the

enzyme was delivered to distend the pancreas. The distended pancreas was

carefully excised and digested in a Dubnoff shaker at 115 cycleslmin for 20

minutes at 37'~. The digested tissue was transferred to cold MI99 (+I 0% CS

and 1 % PIS) solution, washed twice and separated in 10ml Histopaque 1077

(Sigma Chem Co., St. Louis, MO) and 10ml M I 99 (+I % PIS) density gradient at

1000 x g, 10°C for 22 minutes. The islets were collected from the

Histopaque/Ml99 interphase, washed with saline and RPMl 1640. The islets

were then hand picked under the dissecting microscope for encapsulation.

3.1.3 Encapsulation

The encapsulation method (3540,4547) is a modification of the method

of Lim and Sun (35). The rat islets were washed three times with saline and

centrifuged at 120 x g for 2 minutes. The pelleted islets were resuspended in

1.7% (wh) sodium alginate (Kelco Gel LV, Kelco, San Diego, CA) solution. This

mixture was then transferred to a 5 ml syringe containing a small magnetic spin

bar (15 mm x 1.5 mm). The magnetic spiri bar was placed inside the syringe to

ensure complete mixing. An electrostatic droplet generator was used to obtain

small spherical and uniform beads. The beads form due to an electrostatic field

between the needle and the electrode in the calcium lactate solution. The

alginatefislet solution was expelled through a 25G needle into a beaker

containing 100mM calcium lactate solution. The beads were then transferred to a

15ml centrifuge tube. In succession, the capsules were reacted with 0.05% pol)/-

L-lysine (Sigma, M.W. 22,000 to 24,000 Da), 0.17% sodium alginate and 55mM

sodium citrate for 5 minutes each with a 0.9% saline wash between each step.

The islets were then washed three times with RPMl 1640 media and cultured

overnight in a carbon dioxide incubator for m vivo or in vitro studies.

3.1 -4 Transplantation

For each transplant, one thousand encapsulated islets were transferred to

a 15ml centrifuge tube and washed three times with sterile 0.9% saline solution.

Following the last wash, approximately 2ml of islet suspension was left in the

tube for transplantation. Under light ketamine anaesthesia, the islets were

injected into the mouse's intraperitoneal cavity using an 18G catheter

(36,39,40,42,43,91). The opening was closed using surgical staple and the

mouse was allowed to recover under supplemental heat.

3.1.5 Islet Recovery

Under light ketamine anaesthesia, encapsulated rat islets were recovered

from the mice by peritoneal lavage using warm 0.9% saline solution (36,42).

3.1.6 Insulin Content Determination

Insulin was extracted from islets using acid alcohol (92, 100, 104, 105,

107). Encapsulated islets were washed three times with sterile 0.9% saline

solution. Following the last wash, the isiets were suspended in 1mL of acid

alcohol and homogenized for 5 minutes. The homogenate was sonicated for two

30 second intervals. The mixture was incubated overnight at 4%. The following

morning, the supernate was vortexed and centrifuged for 20 minutes at 5000 x g

at 4OC. The supernatant was collected in a separate test tube and incubated at -

20°C. An additional I m l of acid alcohol was added to the pellet followed by

homogenization and sonication. The extract was incubated overnight at 4OC and

the following morning the supernate was vortexed and centrifuged for 20 minutes

at 5000 x g at 4 ' ~ . The supernatants from the two extractions were combined

and the acid alcohol was evaporated under vacuum at 4OC. The pellet was

reconstituted with phosphate buffered saline (PBS) and the samples were then

assayed for insulin.

3.1.7 Glucose Challenge Studies

To assess the viability of both in vivo and in vitro islets, insulin secretion

was measured by exposing islets to increasing concentrations of glucose

(2.8mM, 16.7mM, 16.7mM glucose + O.1mM IBMX) (35, 37, 47). Groups of

twenty islets (nine groupshreatment) were randomly hand-picked under a

dissecting microscope using a 5ml syringe attached to an 18G silicone cannula.

The islets were washed three times to remove any insulin trapped inside the

capsule andincubated at 37OC with 2ml of the glucose containing RPMl 1640

media. Two hours later, 1ml of the media was sampled and assayed for insulin.

3.1.8 Apoptosis

Islets were assayed for apoptosis using the Boehringer Mannheim In Situ

Cell Death Detection Kit (1 16, 1 1 7). Islets were fixed in formalin for 7 days and

mounted on paraffin blocks. Paraffin block and the slides for the apoptosis

assay were made by the Hospital for Sick Children, Department of Pathology.

Sections of the block were mounted on slides and incubated overnight at 60°C.

The following morning the slides were dewaxed in xylene and hydrated using

loo%, 95% and 70% ethanol and distilled water. The slides were then treated

with Proteinase K (20mglmL) in 10 mM TRlSlHCl buffer pH 7.4-8.0 for 30

minutes at room temperature and then washed with distilled water. Following the

wash, the slides were treated with 0.6% H202 in methanol for 30 minutes at room

temperature to remove any endogenous peroxidase and then washed in PBS.

The sections were solubilized with 0.1 % Triton-X in 1 OOmI of 0.1 Oh sodium citrate

for 2 minutes at 4'C and washed with PBS. The area around the sample was

dried and protein block (10% normal sheep serum in PBS) was applied for 10

minutes at room temperature. Following the removal of the protein block,

TUNEL reaction mixture was added for 60 minutes at 37OC. The TUNEL reaction

mixture was further diluted 1.1 with diluting buffer (30mM TRlS pH 7.2 with 150

mM Sodium Cacodylate and ImM CoCI2). The TUNEL reaction mixture is a

combination of two solutions. The first is an enzyme solution containing terminal

deoxynucleotidyl transferase (TdT) and the second is a nucleotide mixture

containing nucleotides conjugated to fluorescein. The sample was then rinsed

with PBS. Converter-peroxidase (POD)- 1 :2 dilution with diluting buffer (1 00mM

TRlSlHCl + 150mM NaCl pH 7.5) was added for 30 minutes at 37OC followed by

a PBS rinse. The slides were then treated with 3,3-diaminobenzidine (DAB)

solution (100ml of 0.05M TrislHCl + 50mg DAB + 50p1 H202) for 4 minutes at

room temperature and then washed in water. Next, the slides were stained with

haematoxylin for 30 seconds, followed by a wash in distilled water. Following the

staining, the slides were dehydrated, rewaxed and mounted with coverslips.

3.1 -9 Computer Assisted Morp hometry

Various fields from the apoptosis assay treated slides were randomly

selected and scanned into the computer. The apoptotic positive nuclei (stained

brown) were marked, counted and expressed as a percentage of the total

number of nuclei (stained blue).

3.1.10 Histology

Islets were fixed in formalin and stained with aldehyde fuchin and

counterstained with neutral red. The aldehyde fuchin (purple stain) stains the

cytoplasm of P cells and neutral red (red stain) stains nuclei. The presence of

purple stain surrounding red stain indicates the presence of viable P cells. The

staining of all samples was performed by the Hospital for Sick Children,

Department of Pathology.

3.2 Experimental Design

3.2. I In Vivo Experimental Design

3.2.1 -1 Transplantation Studies

3.2.1 .I -1 Diabetic Controls

Four mice were made diabetic and maintained for two weeks as untreated

controls.

3 -2.1 .I .2 Transplantation of Unencapsulated Rat Islets

To determine the effects of transplanting unencapsulated rat islets into the

interperitoneal cavity of diabetic C57BU6 mice, each of four mice received 1000

unencapsulated islets. The animals were maintained for at least 2 weeks.

3.2.1.1.3 TransplantationofEmptyCapsules

One thousand empty capsules were transplanted into the peritoneal cavity

of each of 4 diabetic C57BU6 mice. The mice were maintained for at least 2

weeks.

3.2.1.1.4 TransplantationofEncapsulatedRatlslets

Following isolation, the islets were encapsulated and cultured overnight

prior to transplantation. The following morning, the encapsulated rat islets were

3 1

transplanted into the diabetic mice, as described previously, and maintained until

their predetermined recovery period.

3.2.1.2 Plasma Glucose Measurements

Determination of plasma glucose levels was performed using Ames

Glucorneter Elite. Blood samples were taken from the tail vein of the

experimental mice (36,92,93) every day for the first week posttransplantation and

twice a week thereafter.

3.2.1.3 Body Weight Determination

Body weight was determined as an indication of the function of the graft

(37,92) and was performed everyday fo: the first week and twice a week

thereafter.

3.2.1.4 Capsule Recovery

The encapsulated rat islets were recovered from the mice at 3, 7 and 14

days posttransplantation and analyzed far insulin content, insulin secretion,

apoptosis and histology.

3.2.2 Experimental Groups

3.2.2.1 In Vivo Experimental Groups

Altogether, for the in vivo studies, five groups of islets were assessed for

insulin content, insulin secretion, apoptosis and histology. The first group,

32

mencapsulated rat islets, consisted of free rat islets. The second group.

encapsulated rat islets one day in culture, was a sample from the encapsulated

rat islets that were going to be transplanted into the diabetic mice. The

remaining three groups of islets consisted of transplanted islets that were

recovered from the mice at 3, 7 and 14 days posttransplantation, respectiveiy.

3.2.2.2 In Vitro Experimental Groups

Altogether, for the in vifro studies, five groups of islets were assessed for

insulin content, insulin secretion, apoptosis and histology. The first two groups

were the same as above. The remaining three groups were encapsulated islets

cultured in a carbon dioxide incubator for the predetermined time periods of 3, 7

and 14 days, respectiveiy.

3.2.3 Analysis

3.2.3.7 Insulin Content

For each determination, two hundred islets were hand-picked under the

dissecting microscope and homogenized sonicated and assayed for insulin

content. See Figure 2.

3.2.3.2 Insulin Secretion

Islets were assessed for insulin secretcon in response to glucose

challenge and assayed for insulin. See Figdre 3.

3.2.3-3 Apoptosis

For each determination, 200 islets were fixed in forrnalin and assayed for

apoptosis. See Figure 4.

3.2.3.4 Histology

For each determination, 200 islets were fixed in forrnalin and stained with

aldehyde fuchin and neutral red.

Statistics

All values are expressed as mean -+ standard error of the mean (SEM).

Specific statistical analysis used for each study is described in the legend of

each figure. Significance for all statistical analysis is set at ~ ~ 0 . 0 5 .

Figure 1

Experimental Design Flow Chart. The overall summary of the project is

illustrated in figure 1.

Induce Diabetes in C57BU6 Mice I Diabetes is confirmed by 3 consecutive blood glucose measurements above 20mM 11

Rat Islet Isolation Purified islets with an average size of 150 microns were handpicked for encapsulation

Encapsulation (I Ral islets were encapsulated in an alginate-poly-L-lysine-alginale membrane 11

I

Transplantation 1000 encapsulated rat islels were transplanted inlraperiloneally

into each experimental animal and recovered at 3,7 and 14 days postlransplanlalion

I

In Vifro Studies Encapsulated rat islets were cultured at

3 7 O C I 5% C02 for 3 , 7 and 14 days

h

I I I

Insulin Secretion Using an in s lu cell death detection kit Islets were incubated with glucose concenlralions

of 2.8mM, 16.7mM and 16.7mM + IBMX

Insulin Content Insulin was extracted from islets using

an acid alcohol method L

Figure 2

insulin Content Flow Chart. A summary of the insulin extraction protocol.

Insulin Content

Experimental Group L

A

L

Handpick 200 Islets L

Homogenize, Son icate,

Centrifuge to obtain Insulin Extract

m

RIA samples for Insulin Content

v

Figure 3

Insulin Secretion Flow Chart. A summary of the insulin secretion protocol.

Insulin Secretion

Experimental Group r Handpick 9 groups

of 20 islets n Challenge islets with 2.8mM glucose, 16.7rnM glucose or 16.7mM glucose

+ O.lmM IBMX

I

2.8mM Glucose 16.7mM Glucose 3 groups of 20 islets

I t

16.7mM Glucose + O.lmM IBMX 3 groups of 20 islets

I

RIA for insulin secretion €I

Figure 4

Apoptosis Flow Chart. A summary of the apoptosis protocol.

Apoptosis Assay

Experimental Group .J L

I A

Handpick 200 Islets L L

m

7

Fix islets in formalin and process on a

paraffin block

f I

Apoptosis Detection Kit 2

I

Computer Assisted Morphometry Quantification of Apoptotic Nuclei

L

CHAPTER 4

4. RESULTS

4.1. Transplantation Studies

4.1.1 Diabetic Control

This experiment was performed to determine the reliability of the STZ-

induced diabetic mouse model (Figure 5). Diabetes was induced in four

C57BU6 mice. The mice were observed for a 16-day period over which blood

glucose and body weight were monitored daily. There was a significant increase

(p<0.0004) in the blood glucose levels of all mice seven days after the induction

of diabetes as compared to pre-diabetic blood glucose levels. In particular, pre-

diabetic blood glucose values of the mice were between 5 to IOrnrnol/L, whereas

after the induction of diabetes the mice maintained hyperglycemic blood glucose

levels (23.2-30.0mmolfL) for the entire 15-day observation period. As well,

compared to pre-diabetic values (1 8 - X g ) , the induction of diabetes was

associated with a decrease in body weight of the experimental animals over the

observation period by 2-3 grams (Figure 1 1 ).

Figure 5

Diabetes was induced in four mice using 185rng of ST2 I kg body weight. Blood

glucose was measured one week after the induction of diabetes. There was a

significant increase (p<0.0004) in blood g l~cose levels of all mice (>20mmol:L)

after the induction of diabetes as compared to pre-diabetic blood glucose levels

(5-1OmmollL). As illustrated in the graph, diabetes was maintained for the entire

16 day observation period. Statistical analysis was performed using a two-

sample f-test.

Blood Glucose Profiles of Diabetic Control Mice

First blood glucose measurement, 1 week post* induction of diabetes

+ Control 2

+Control 3

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Time (Days)

4.1.2 Transplantation of Unencapsulated Rat Islets

One thousand unencapsulated rat islets were transplanted into the

intraperitoneal cavity of four C57BU6 mice (Figure 6). As seen in figure 6, the

transplanted unencapsulated rat islets restored normogiycemia (5-1 OmmollL

blood glucose) by day three posttransplantation. This effect was transient and

lasted for only four days. By day seven posttransplantation blood glucose had

returned to hyperglycemic levels (>20mmoVL) in all four transplants. All mice

remained hyperglycemic (21.5-30.0mmollL) for the remainder of the 16 day

observation period.

Figure 6

The graph illustrates that unencapsulated rat islets were able to lower blood

glucose lev& by day 3 posttransplantation. At day 7 posttransplantation there

was an increase in blood glucose levels of the transplanted mice. The data

shows that transplanted unencapsulated rat islets were not able to maintain

nonoglycemia after day 6 posttransplantation.

Blood Glucose Profiles of Diabetic Mice Transplanted with Unencapsulated Rat Islets (URI)

Transplantation A

-+-Recipient One

-W- Recipient Two

4 Reclplent Three

+Recipient Four

6 7 8 9

Time (days)

4.1 -3 Transplantation of Empty Capsules

One thousand empty capsules were transplanted into the intraperitoneal

cavity of four diabetic C57BU6 mice (Figure 7). This experiment was performed

to determine the effects of alginate-poly-L-lysine-alginate (APA) capsules on the

diabetic mice. The transplanted mice were observed over a 16-day period

during which blood glucose and body weight were measured. All four rnic.3

remained hyperglycemic (23.8-30.0mmollL) for the duration of the study. As with

the diabetic controls, the induction of diabetes caused a 2-39 decrease in body

weight in the experimental mice compared to pretransplantation values (Figure

11). This decrease in body weight was maintained throughout the study period.

Figure 7

One thousand empty capsules were transplanted into the intraperitoneal cavity of

each of four diabetic mice. The graph shows there was no change in blood

glucose levels of the mice after the empty capsule transplantation.

Blood Glucose Profiles of Diabetic Mice Transplanted with Empty Capsules

Transplantation of 1000 Empty Capsules

-+- Recipient 1

+Recipient 2

+ Recipient 3

36 Recipient 4

- 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time (days)

4.1.4 Transplantation of Encapsulated Rat Islets

One thousand encapsulated rat islets were transplanted into six diabetic

C57BU6 mice and observed for three days (Figure 8). There was a significant

decrease (pc0.0001) in the blood glucose levels of all mice by day one

posttransplantation. All six mice became normoglycemic (4.5-9.2mmollL) by 24

hours posttransplantation and maintained norrnoglycemia throughout the

observation period of three days. There was an approximate l g increase in

body weight of the transplanted mice throughout this period (18-219).

One thousand encapsulated rat islets were transplanted into another

seven diabetic C57BU6 mice and obsewed for seven days (Figure 9). Similar to

the three day study, all seven mice achieved normoglycemia (4.6-9.8mmollL) by

day one posttransplantation (p<0.0001). Normoglycemia was maintained in all

mice throughout the seven days. There was a 2-39 increase in body weight of

the transplanted mice from day one to day seven.

As with the seven day study, 1000 encapsulated rat islets were

transplanted into another six diabetic C57BU6 mice and observed for 14 days

(Figure 10). All six mice became normoglycemic (4.4-9.9rnmollL) by 24 hours

posttransplantation (p<0.0001). Normoglycernia was maintained throughout the

14 day observation period. As above, there was a 2-39 increase in body weight

in the experimental mice (Figure 11). As compared to diabetic control mice and

mice transplanted with empty capsules, there was a significant increase

(p<0.0001) in body weight of mice transplanted with encapsulated rat islets for

14 days.

At sacrifice, the peritoneal cavity and the recovered capsules were

examined for all transplanted mice. All 19 mice had normal peritoneal cavities

with no grossly observable foreign tissue reactions, sterile adhesions, or

peritonitis. Capsules recovered were free of overgrowth (Figure 12). Between

40% and 60% of the transplanted capsules were recovered from each

experimental animai.

Figure 8

The figure illustrates that all mice became normoglycemic (5-IOmmollL) by day

one posttransplantation and were able to maintain normoglycernia throughout

the three day study period. There was a significant decrease (p<0.0001) in

blood glucose levels of all mice 24 hours after transplantation as compared to

pre-transplantation levels. Statistical analysis was performed using one-way

analysis of variance (ANOVA) with repeated measure followed by a two-sample

t-test with Bonferroni correction.

Blood Glucose Profiles of Diabetic Mice Transplanted with Encapsulated Rat Islets (ERI) for 3 Days

\ Transplantation of 1000 ERI

. - .

+Mouse 1

-R- Mouse 2

+Mouse 3

+Mouse 4

+Mouse 5

+Mouse 6

1

Time (days)

Figure 9

The figure shows there was a significant decrease (p~O.0001) in blood glucose

levels of ail mice by day one posttransplantation with 1000 ERI. The mice

maintained norrnoglycemia (5-IOmmollL) for the entire seven study. Statistical

analysis was performed using one-way analysis of variance (ANOVA) with

repeated measure followed by a two-sample t-test with Bonferroni correction.

Blood Glucose Profiles of Diabetic Mice Transplanted with Encapsulated Rat Islets (ERI) for 7 Days

Transplantation of 3000 ERI

- -- - - -

-+Mouse 1

--C- Mouse 2

-4h- Mouse 3

+Mouse 4

+Mouse 5

--f- Mouse 6

+Mouse 7

2 3 4

Time (days)

Figure 10

The figure illustrates that all mice became normoglycernic by day one

posttransplantation. There was a significant decrease (p<0.0001) in the blood

glucose levels of all mice as compared to pre-transplantation levels. Statistical

analysis was performed using one-way analysis of variance (ANOVA) with

repeated measure followed by a two-sample t-test with Bonferroni correction.

Blood Glucose Profiles of Diabetic Mice Transplanted with Encapsulated Rat Islets (ERI) for 14 Days

Transplantation of 1000 ERI

. . ... >.. . . .- -. .-

+Mouse 1

+Mouse 2

15 Mouse 3

Jt- Mouse 4

+ Mouse 5

+Mouse 6 " . - . . - - .

Time (days)

Figure 11

The figure shows a decrease in body weight of diabetic control mice and mice

transplanted with empty capsules. However, the figure also illustrates an

increase in body weight of mice transplanted with encapsulated rat islets for 14

days. There was a significant increase (p<0.0001) in body weight of mice

transplanted with encapsulated rat islets as compared to both diabetic control

mice and mice transplanted with empty capsules at day 14. Statistical analysis

was performed using one-way analysis of variance (ANOVA) with repeated

measure followed by a two-sample t-test with Bonferroni correction.

Body Weinht Determination of Diabetic Control Mice, Diabetic Mice Transplanted with E m ~ t v Capsules and Diabetic Mice Transplanted

with Enca~sulated Rat Islets [ERI) for 14 Days

+ Dlabetic Control

+Empty Capsules

--t- ERI Transplanted for I

I Days

6 7 8

Time (days)

Figure 12

Photomicrograph of Encapsulated Rat Islets Recovered at 14 Days

Posttransplantation. The figure illustrates that the capsules were intact after 14

days in vivo and there was no fibrotic overgrowth on the capsular surface. (LM

130X)

Figure 12

Photomicrograph of Encapsulated Rat Islets Recovered at 14 Days

Posttransplantation. The figure illustrates that the capsules were intact after 14

days in vivo and there was no fibrotic overgrowth on the capsular surface. (LM

l3OX)

4.2 Insulin Content

4.2.1 Cultured Islets

For each experimental group, 200 islets were homogenized, sonicated

and assayed for insulin (Figure 1 3a). Unencapsulated rat islets were cultured

overnight prior to being extracted for insulin. Encapsulated rat islets one day in

culture were a sample from the encapsulated islets that were scheduled to be

transplanted. Insulin was also extracted from the remaining three groups which

represent islets cultured for 3, 7 and 14 days, respectively. The graph shows

that there was less insulin stored in the encapsulated islets over time. There was

no significant difference (pr0.05) in the amount of insulin stored in the

unencapsulated rat islets (35.1 k 5.1 pg) as compared to the encapsulated rat

islets one day in culture (31 -4 + 2.6pg). Comparing freshly isolated

unencapsulated rat islets to the cultured islets, there seemed to be a significant

decrease in the amount of insulin stored longer the islets were cultured. P'

3, 7 and 14 days in vitro, the cultured islets were found to contain 28.9 f 2.8pg

(pc0.01), 25.1 f 2.6pg (p<0.0001) and 16.1 t 2.4~9 (pc0.0001) of insulin,

respectively.

4.2.2 Recovered Islets

As above, 200 recovered islets were homogenized, sonicated and

assayed for insulin (Figure 13b). As with the cultured islets, the recovered islets

appeared to store less insulin over time. There was no significant difference

(p>0.05) in the amount of insulin stored between unencapsulated rat islets (35.1

k 5.1 pg) and encapsulated rat islets one day in culture (31 -4 + 2.6). However, as

compared to the unencapsulated rat islets (35.1 + 5.1pg), there was a significant

decrease (p<0.0001) in the amount of insulin stored in the recovered islets at 3,

7 and 14 days, 16.5 k 1.2pg, 15.1 r 2.9pg and 1 1.5 + 2.3pg, respectively. There

was no significant difference ( ~ ~ 0 . 2 0 ) in the amount of insulin stored between

the islets recovered at 3 days and 7 days posttransplantation. However, there

was a significant difference ( ~ ~ 0 . 0 2 ) in the amount of insulin stored between the

islets recovered at 3 days and those recovered at 14 days posttransplantation.

4.2.3 Comparison of Recovered and Cultured Islets

The first two groups, unencapsulated rat islets and encapsulated rat islets

one day in culture were of the same batch with both recovered and cultured

islets. As stated earlier, there was no significant difference (p>0.05) in the

amount of insulin stored between the two groups. Comparing the recovered and

cultured islets at their respective time periods (3, 7, 14 days) it appeared that all

cultured islets contained significantly more insulin than the recovered islets

(p~0.002, pc0.005, pc0.04, respectively) (Figure 13c). Islets cultured for 3, 7

and 14 days contained 28.9 + 2.8~9, 25.1 + 2.6pg and 16.1 k 2.4pg of insulin,

respectively, while the insulin content of recovered islets at 3, 7 and 14 days was

determined to be 16.5 + 1.2pg, 15.1 + 2.9pg and 11 -5 f 2.3pg1 respectively.

Figure 13a

The figure illustrates a decrease in insulin content in the in vitro cultured islets as

compared to unencapsulated rat islets over time. Means without a common

letter are significantly different (pc0.05). Statistical analysis was performed using

one-way analysis of variance (ANOVA) with repeated measure followed by a

two-sample t-test with Bonferroni correction.

Figure 13b

The figure shows a decrease in insulin content of the recovered rat islets as

compared to onencapsulated rat islets over time. Means without a common

letter are significantly different (~~0.05). Statistical analysis was performed using

one-way analysis of variance (ANOVA) with repeated measure followed by a

two-sample t-test with Bonferroni correction.

Insulin Content. Recovered lslets as Compared to Untransplanted Controls

Unencapsulated Encapsulated Encapsulated Encapsulated Rat Islets (n=5) Rat Islets 1 Day Rat Islets Rat lslets

in Culture (n=5) Recovered at 3 Recovered at 7 Days (n=4) Days (n=4)

Encapsulated Rat lslets

Recovered at 14 Days (n=4)

Figure 13c

The figure illustrates the insulin content of in vitro cultured islets and recovered

islets at 3, 7 and 14 days. The graph shows that all cultured islets contained

significantly more (pc0.04) insulin than the recovered islets at each respectiw

time period (3. 7 and 14 days). Statistical analysis was performed using two-

sample f-tests.

Insulin Secretion

4.3.1 Cultured Islets

As previously mentioned, the five in vitro experimental groups were

assessed for insulin secretion in response to glucose challenge and assayed for

insulin (Figure 14a). The graph shows that as compared to the unencapsulated

rat islets, there was less insulin secreted by the cultured islets over time (Figure

14a). The unencapsulated rat islets secreted more insulin for each respective

glucose concentration than the cultured islets. Unencapsulated rat islets

secreted 0.48 + 0.1 4qg/islets/2hr, 3.02 + 0.23qglislets12hr and 4.09 +

O.PO-rlglislets/2hr of insulin for the 2.8mM, 16.7mM and 16.7mM glucose +

O.lmM IBMX, respectively while the islets cultured for 14 days secreted 0.09 t

0.17qglislets/2hr, 0.52 + 0.12~glislets/2hr and 1 -66 k 0.31 ~glisletsl2hr of insulin,

respectively, for the same glucose concentrations. At the 2.8mM glucose

concentration, there was no significant decrease ( ~ ~ 0 . 1 ) in insulin secretion

between the unencapsulated rat islets and the remaining four groups. At the

16.7mM glucose concentration, there was a significant decrease in insulin

secretion between the unencapsulated rat islets and islets cultured for 3, 7 and

14 days (pc0.005, p<0.001, pe0.001, respectively). At l6.7mM glucose +

0.01 mM IBMX there was a there was a significant decrease (pc0.001) in insulin

secretion between the unencapsulated rat islets and islets cultured for 3, 7 and

14 days. In general, islets challenged with the higher glucose concentrations,

16.7mM and 16.7mM + O.1mM IBMX, released 3 to 6 times, and 6 to 8 times

more insulin, respectively, as compared to islets exposed to the 2.8mM glucose

concentration.

4.3.2 Recovered Islets

The recovered islets were challenged with different glucose

concentrations and assayed for insulin secretion (Figure 14b). As with the

cultured islets, there was less insulin secreted by the recovered islets over time.

Unencapsulated rat islets secreted 0.48 f 0.1 4qg/islets/2hr1 3.02 +

0.23qgfislets12hr and 4.09 k 0.20qg/islets/2hr of insulin for the 2.8mM. 16.7mM

and 16.7mM glucose + O.lmM IBMX, respectively while the islets recovered at

14 days secreted 0.22 k 0.l5~g/isIets/2hrl 1.22 k O.i8rlg/isletsQhr and 2.02 k

0.22qglislets/2hr of insulin, respectively, for the same glucose concentrations. At

the 2.8mM glucose concentration, there was no significant decrease (p>0.1) in

insulin secretion between the unencapsulafed rat islets and the remaining four

grcups. At the l6.7rnM glucose concentration, there was a significant decrease

in insulin secretion between the unencapsolated rat islets and islets cultured for

3, 7 and 14 days (pc0.02, pe0.005, p<0.001, respectively). At 16.7mM glucose

+ 0-OlmM IBMX there was a significant decrease in insulin secretion between

the unencapsulafed rat islets and islets cultured for 3, 7 and 14 days (pc0.01,

p<0-001, p~0.001, respectively). In general, islets challenged with the higher

glucose concentrations, 16.7mM and 16.7mM + O.lmM IBMX, released 3 to 6

times, and 6 to 8 times more insulin, respectively, as compared to islets exposed

to the 2.8rnM glucose concentration.

4.3.3 Comparison of Recovered and Cultured Islets

it is evident from figures 14a and 14b that the recovered islets secreted

more insulin over time for each respective glucose concentration than did the

cultured islets.

Figure 14a

The figure illustrates the amount of insulin secreted by in vjtro cultured islets over

time using 2.8mM, 16.7mM and 16.7mM + O.?mM IBMX. The graph shows no

significant decrease (pzO.1) in insulin secretion at 2.8mM between the islets

cultured for 3, 7 and 14 days as compared to unencapsulated rat islets. At

l6.7mM and 16.7mM + 0.1 rnM IBMX, there was a significant decrease (pc0.005)

in insulin secretion between the islets cultured for 3, 7 and 14 days as compared

to unencapsulated rat islets. Statistical analysis was performed using one-way

analysis of variance (ANOVA) with repeated measure followed by a two-samp!e

t-test with Bonferroni correction.

Glucose Challenge, In Vitro Cultured Islets

- -. . . . . - -

Unencapsulated Rat Islets, n=6

.Encapsulated Rat Islets 1 Day in Culture, n=7

OEncapsulated Rat Islets 3 Days In Vitro, n=5

OEncapsulated Rat Islets 7 Days In Vitro, n=6

Encapsulated Rat Islets 14 Days In Vitro, n=6

Glucose (mM)

Figure 14b

The figure illustrates the amount of insulin secreted by recovered islets over time

using 2.8mM, 16.7mM and 16.7mM + O.lmM IBMX. The graph shows no

significant decrease (p>0.1) in insulin secretion at 2.8mM between the recovered

islets at 3, 7 and 14 days posttransplantation as compared to onencapsulated rat

islets. At l6.7mM and l6.7mM + 0.1 mM IBMX, there was a significant decrease

(pc0.02) in insulin secretion between the recovered islets at 3. 7 and 14 days as

compared to onencapsulated rat islets. Statistical analysis was performed using

one-way analysis of variance (ANOVA) and two-sample t-tests.

Glucose Challenne, Recovered lslets as Compared to Untransplanted Controls

ISI Unencapsulated Rat Islets, n=6

Encapsulated Rat lslets 1 Day in Culture, n=7

OEncapsulated Rat lslets Recovered at Day 3, n=5

0 Encapsulated Rat lslets Recovered at Day 7, n=7

IEncapsulated Rat lslets Recovered at Day 14, n=6 . . . -

Glucose (mM)

Apoptosis

4.4.1 Cultured Islets

For each in vitro experimental group, 200 islets were fixed in forrnalin and

assayed for apoptosis (Figure 15a). There was a significant increase (pc0.01) in

the amount of apoptosis occurring in the encapsulated rat islets one day in

culture (23.2 k 2.0% apoptotic nuclei) as compared to unencapsulated rat islets

(15.9 f 2.3%). In addition, it appears that the amount of apoptotic nuclei remain

stable over time. At 3, 7 and 14 days in vitro culture. the amount of apoptosis

occurring was 25.2 + 1.0%, 28.9 t 2.2% and 25.4 + 4.3%, respectively. All

cultured islets had apoptotic rates significantly higher (pc0.01) than the

unencapsulated rat islets (1 5.9 k 2.3%).

4.4.2 Recovered Islets

As above, there were five groups of rat islets analyzed for apoptosis

(Figure 15b). The graph shows a decrease in the extent of apoptosis occurring in

the recovered islets over time. As compared to unencapsulated rat islets (1 5.9 +

2.3% apoptotic nuclei), there was a significant increase (p<0.01) in the amount of

apoptotic nuclei in the encapsulated rat islets one day in culture (23.2 -+ 2.0%).

However, there was less apoptosis occurring in the recovered islets the longer

they remained in vivo. At 3, 7 and 14 days posttransplantation, the amount of

apoptosis occurring in the retrieved islets was reduced to 21.1 + 2.0%, 18.9 + 1.9% and 15.1 k 0.9, respectively. In fact, there was no significant difference

(pNI.3) in the rate of apoptosis occurring in the islets recovered at 14 days

posttransplantation (15.1 + 0.9%) as compared to the unencapsulated rat islets

(15.9 f 2.3%).

4.4.3 Comparison of Recovered and Cultured Islets

From figure 15c it can be seen that there was a significant difference

(pc0.01) in the amount of apoptosis occurring in the recovered versus the in vitro

cultured islets for each respective time period. With recovered islets there

appeared to be a decrease in the amount of apoptosis occurring over time while

there was an increase in the amount of apoptosis occurring in the cultured islets

over time. More specifically, recovered islets contained 21.1 + 2.0%. 18.9 k

1.9% and 1 5.1 + 0.9% apoptotic nuclei at 3, 7 and 14 days posttransplantation,

respectively, while cultured islets contained 25.2 + 1.0%, 28.9 k 2.2% and 25.4 k

4.3% apoptotic nuclei, after 3, 7, and 14 days in culture, respectively.

Figure 15a

The figure illustrates the rate of apoptosis of in vitro cultured islets over time.

Means without a common letter are significantly different (pe0.05). Statistical

analysis was performed using one-way analysis of variance (ANOVA) with

repeated measure followed by a two-sample t-test with Bonferroni correction.

Figure 15b

The figure illustrates the rate of apoptosis of islets recovered at 3, 7 and 14 days

posttransplantation. Means without a common letter are significantly different

(pe0.05). Statistical analysis was performed using one-way analysis of variance

(ANOVA) with repeated measure followed by a two-sample t-test with Bonferroni

correction.

Figure 15c

The figure illustrates the percentage of apoptotic nuclei of in vifro cultured islets

and recovered islets at 3, 7 and 14 days. The graph shows that all cultured islets

had significantly higher (pc0.0001) rates of apoptosis than the recovered islets at

each respective time period (3, 7 and 14 days). Statistical analysis was

performed using two-sample t-tests.

Apoptosis: In Vitro Cultured Versus Recovered Rat Islets

, - - -

Bl In Vitro Cultured Islets, n=4

I . Recovered Rat Islets, n=4

3 Days 7 Days 14 Days

4.5 Electron Microscor>y

4-51 Islets Recovered at 7 Days Posttransplantation

Ultrastructural details of both recovered and in vitro cultured islets were

obtained using electron microscopy. Islets recovered at 7 da.).

posttransplantation (Figure 16) revealed apoptotic, necrotic and normal nuclei.

Early stages of apoptosis were identified by the condensed chromatin inside the

nuclei (Figure 16a) as compared to normal nuclei (Figure 16b). In addition,

electron microscopy also revealed what appeared to be irregular euchrornatic

nuclei, a characteristic of necrotic nuclei (Figure 16c). All electron microscopy

was performed by Microscopy Imaging Laboratory, University of Toronto, Faculty

of Medicine.

4.5.2 In Vifro islets Cultured for 7 Days

Electron microscopy of in vitro islets cultured for 7 days revealed

apoptotic, normal and necrotic nuclei (Figure 17). As above, apoptotic cells weye

identified in figure 17a by the condensed chromatin inside the nuclei. in addition,

viable cells are also illustrated in figure 17a, as indicated by the numerous

mitochondrion and insulin granules as well as non-condensed chromatin (Figure

17a). Furthermore, an irregular euchromatic nuclei, a characteristic of necrotic

cell death, can be seen in figure 17b.

Figure 16a.

Apoptotic P cell of the recovered rat islets at 7 days posttransplantation.

Electron microscopy of a recovered islets p cell showing condensed chromatin, a

characteristic of apoptosis. (EM 1 1220X)

Figure 16b.

Normal P cells of recovered rat islets at 7 days posttransplantation. The non-

condensed chromatin in addition to numerous insulin granules within the

cytoplasm of the P cells is indicative of viable cells. (EM 7820X)

Figure 16c.

Necrotic p cell of recovered rat islets at 7 days posttransplantation. The irregular

euchromatic nuclei illustrated is a characteristic of necrotic cell death. Note the

minimal viable insulin granules within the cytoplasm. (EM l36OOX)

Figure 17a.

Apoptotic and viable P cells in 7 day in vitro cultured rat islets. The condensed

chromatin illustrated in figure 17a is indicative of apoptotic nuclei (see thick black

arrow). However, the numerous insulin granules and mitochondrion also shown

in figure 17a is indicative of viable P cells (thin black arrow). (EM 561 OX)

Figure 17b.

Necrotic p cell of 7 days in vitro cultured rat islets. The figure shows an irregular

shaped nucleus and disrupted cytoplasm containing very few insulin granules.

This type of cell death is characteristic of necrosis. (EM 13600X)

4.6 Histoloclv

A photomicrograph of encapsulated islets stained with aldehyde fuschin

and counterstained with neutral red is seen in Figure 18. Encapsulated islets

one day in culture (Figure 18a), islets recovered at 14 days posttransplantation

(Figure 18b) and in vitro islets cultured for 14 days (Figure 18c) were assessed

for viable P cells. Aldehyde fuschin (purple stain) stains the cytoplasm of P cells

!n co- while neutral red stains all viable nuclei red. The presence of purple sta'

localized with the red stain reveals the presence of viable P cells. As seen in all

three photomicrographs, the prominent presence of purple and red stains

indicates strong evidence for viable P cells.

Figure 18a.

Encapsulated rat islets one day in culture stained with aldehyde fuchin and

neutral red. The presence of purple co-localized with the red stain indicates the

presence of viable P cells. The photomicrograph shows that the encapsulated

rat islets one day in culture contain many viable P cells. (LM 640X)

Figure 18b.

Encapsulated rat islets recovered at 14 days posttransplantation stained with

aldehyde fuchin and neutral red. As previously mentioned, the presence of

purple co-localized with the red stain indicates the presence of viable P cells.

Figure 18b illustrates that the recovered islets still contain many viable P cells.

(LM 640)

Figure 18c.

Encapsulated rat islets in vitro cultured for 14 days stained with aldehyde fuchin

and neutral red. The figure illustrates the presence of many viable P cells as

indicated by the co-localized purple and red stains. (LM 640)

CHAPTER 5

5. DISCUSSION

In this study the vulnerability of encapsulated islets transplanted into

diabetic mice over a 3, 7 and 14 day time period was investigated. There are

many changes that occur in the islets in the period immediately following

transplantation. To date, very little information about such changes is available.

The purpose of this study was to examine the early fate of encapsulated islets

and determine the dynamic changes that occur in the immediate

posttransplantation period. In addition, the early fate of encapsulated islets

cultured in vitro was also analyzed to determine the effects of culture conditions

on the islets.

5. I Transplantation Studies

Streptozotocin was used to produce a chemically induced model of type 1

diabetes by destroying the P cells of the islets of Langerhans. The

streptozotocin induced diabetic mouse model proved to be effective as

demonstrated in the diabetic control experiment (Figure 5). The diabetic control

mice showed a decrease in body weight during the observation period, as also

previously reported (94). The decrease in body weight may be attributed to the

catabolic effects due to the lack of insulin.

Transplantation of 1 000 unencapsulated rat islets into the intraperitoneal

cavity of diabetic mice proved to be an ineffective way of treating diabetes. The

unencapsulated islets were only able to transiently produce normoglycemia

(Figure 6). The transient normoglycemia observed with unencapsulated rat islets

transplanted in diabetic mice correlates with reports (45,53.95) of

immunorejection in other studies. It is likely that, by day seven, the

unencapsulated islets were rejected by the host, thus compromising circulating

insulin levels and reinstating hyperglycemia.

Empty capsule transplantation studies revealed that the microcapsules

themselves do not affect the recipient's blood glucose levels. This finding

correlates with other reports (36,54) according to which empty capsules have no

effects on normalizing blood glucose levels of diabetic animals.

The transplantation of 1000 encapsulated rat islets into diabetic mice

allowed blood glucose levels to achieve norrnoglycemia for the entire 14 day

observation period (Figure 10). The islets were able to function for a longer

period of time than the unencapsulated rat islets because of the

immunoprotection accorded by the alginate-poly-L-lysine-alginate microcapsule.

The semipermeable nature of the capsule allows nutrients, hormones and gases

to pass through (cut-off 60,000M.W.), but is impermeable to immunoglobulins

(-1 00,000M.W.) and other larger molecules (54). The smaller capsule's

sphericity and smoothness also contribute to its lack of contact irritation,

therefore protecting the transplanted islets and capsules from recipient's immune

system (54). It has been shown that cellular overgrowth appears when the

capsular surface compatibility is compromised (45). Capsular overgrowth has

long been considered a major cause of graft failure (96). Moreover, it has been

previously reported that if the microcapsule is not biocompatible, the membrane

develops fibrotic overgrowth (97,98). Microcapsules recovered at 3, 7 and 14

days posttransplantation were free of overgrowth indicating that the capsule was

effective in providing irnmunoisolation for the transplanted tissue.

The body weight of the transplanted mice increased (Figure 11) during all

three time periods. Correlating with previous reports (92,94.95,99,100), the

increase in body weight in the experimental animals may be attributed to the

anabolic effects of insulin secreted from the p cells of the transplanted islets.

Increased insulin levels allowed for: the uptake and storage of glucose by the

liver and muscle; increased synthesis and storage of lipids in adipocytes;

increased protein synthesis; and decreased catabolism of glycogen, fats and

proteins (4). In addition to increased body weight, there was an increase in the

activity levels of the mice and better grooming patterns following transplantation

(36) -

5.2 Insulin Content

Insulin content studies reveal ed there was no significant difference in the

p cell insulin content between unencapsulated rat islets and encapsulated islets

one day in culture. As previously reported (101,102), the study shows that

encapsulation does not extensively damage the P cells and thus does not

significantly change the insulin stores between unencapsulated rat islets and

encapsulated islets one day in culture.

107

There was a significant decrease in the insulin content between the

unencapsulated rat islets and the recovered islets over time (Figure 13b). It is

likely that the decreased insulin stores in the recovered islets may be attributed

to the effects of the hyperglycemic environment. Being placed in a

hyperglycemic environment, there was a continual demand for P cells to secrete

significant amounts of insulin to normalize blood glucose levels. It is likely that

because there was an increased demand for insulin, less emphasis was placed

on insulin storage while insulin secretion increased, thus resulting in decreased

insulin content. These results correlate with previous studies (j 03-1 06)

indicating a decrease in insulin content in the recovered islets over time. As wsll,

it is possible that some degree of p cell death could be responsible for

decreased insulin content over time.

The in vitro cultured islets also stored less insulin than the

unencapsulated rat islets (Figure 13a). In vitro islets were cultured with media

containing sub-basal glucose concentration (2.8mM). This very low glucose

concentration would not lead to glucose stimulated insulin secretion. It is likely

that the decrease in insulin content in the in vitro cultured islets was due to basal

insulin release that occurs irrespective of glucose concentration. Recent studiec

have reported that glucose dose-dependently increases the percentage of P cells

actively involved in insulin biosynthesis (31). Low glucose levels in the culture

media would likely cause a decrease in the number of active p cells. Fewer

active p cells would then contribute to the overall decreased insulin biosynthesis

in the cultured islets (31 ,I 07). It is likely that decreased insulin biosynthesis, with

continuous basal insulin secretion by the islets, results in the depletion of insulin

stores over time (Figure 13a). P cell death could also be responsible for

decreased insulin content over time. It is believed that glucose promotes the

survival of p cells by activating synthesis of proteins that suppress apoptosis

(31,107). Absence of physiological concentrations of glucose in in vitro cultured

islets leads to decreased inhibition of proteins that suppress apoptosis and

therefore increase beta-cell death (108). Therefore the in viiro islets over time,

being exposed to sub-physiologic glucose concentrations, may have higher rates

of apoptosis. In addition, it has been reported that in normoglycemic animals

(109,110) there is a close and highly significant correlation between pancreatic

insulin content and p cell volume, and it is likely that this is also the case for

transplanted islets (1 09,111). Given that insulin content and P cell volume are

correlated, the decreasing insulin content in the cultured islets over time may

correspond with a decrease in the number of P cells and thus an increase in P

cell death.

5.3 Insulin Secretion

As demonstrated in the glucose challenge study, recovered rat islets were

still capable of glucose stimulated insulin secretion (Figure 14b). There was no

significant difference in the amount of insulin secreted by unencapsulated rat

islets and encapsulated islets one day in culture. This finding indicates that

encapsulation did not extensively damage the P cells and thus does not

significantly change insulin secretion between the two groups of islets. However,

this finding is contradictory to results by Sandler et al (101) who reported a

significant decrease in the amount of insulin secreted by encapsulated islets, in

comparison to unencapsulated rat islets one day in culture. The difference in

findings may be explained by a different encapsulation technique and membrane

quality. Although Sandier's capsules are chemically composed of alginate-poly-

L-lysine-alginate, their average diameter was 0.7mm instead of 0.25-0.35mm,

and the alginate beads were formed by pressing the alginate-islet solution

through a 22-gauge syringe as opposed to being formed by an electrostatic

droplet generator.

There was a decrease in the amount of insulin secreted by the recovered

islets as compared to the unencapsulated rat islets (Figure 14a). This decrease

may be attributed to islet exhaustion as previously reported (1 12). As mentioned

above, there is a continuous demand for islets to secrete insulin in a

hyperglycemic environment. As a result, it is possible that the islets become

fatigued and are unable to secrete large quantities of insulin in a short period of

time (2 hours) in response to glucose stimulation. In addition, it is likely that

there is some degree of P cell death that occurs in the islets over time, resulting

in decreased insulin secretion. However, as mentioned above, in in vitro cultures

it is believed that glucose promotes the survival of P cells by activating synthesis

of proteins that suppress apoptosis (31,107). Thus, high glucose concentrations

are actually beneficial for P cell survival. Recruitment of P cells into insulin

biosynthesis is glucose concentration dependent and also attributed to an

intercellular heterogeneity in the metabolic threshold for glucose metabolism

(1 13). Prolonged exposure to 1 OmM glucose maintains the majority of P cells in

an active state and therefore the rate of apoptosis and cell death is low. From

these reports, it is possible that the same kinetics also apply to in vivo cultures.

It is probable that the increased glucose concentration in the hyperglycemic

environment of the diabetic animals is advantageous for the transplanted P cells.

From this assumption, it is likely that, if there is any P cell death, it is not likely

due to apoptosis.

As compared to the mencapsulated rat islets, there was a decrease in the

amount of insulin secreted over time in the in vitro cultured islets (Figure 14a).

This finding correlates with other studies that have shown a decrease in insulin

secretion by in vitro islets cultured over time (101). It is conceivable that culture

conditions do not favour insulin biosynthesis and thus result in less secretion. In

fact it has been reported (31) that lower glucose concentration decreases the

number of active p cells and thus insulin biosynthesis. In addition, it has been

shown that when the glucose concentration of media was reduced from 10mM to

3mM, the percentage of active P cells dramatically decreased (1 14,115) and a

parallel increase was seen in the percentage of apoptotic P cells (31). Thus, it is

likely that the decrease in insulin secretion in the in vitro cultured islets may be

attributed to a smaller number of active P cells producing insulin and to

increased p cell death, in particular apoptosis.

5.4 Apoptosis

As compared to unencapsulated rat islets, there was a significant increase

in the percentage of apoptotic nuclei in encapsulated rat islets one day in culture

(Figure 15b). The increased apoptosis in the encapsulated rat islets may be

attributed to the physical and chemical trauma experienced by the islets during

the encapsulation process. It is likely that the absence of glucose during

encapsulation may lead to decreased inhibition of proteins that suppress

apoptosis and therefore increase beta-cell death (31,108). There was a

decrease in the amount of apoptotic nuclei between the encapsulated islets one

day in culture and the recovered islets (Figure 15b). The decrease in apoptosis

was not significant in the 3 day time period, but by 14 days posttransplantation,

there was a substantial drop in the percentage of apoptotic nuclei present.

Recent studies have shown that p cell apoptosis peaks at 3 days

posttransplantation and decreases by 14 days posttransplantation (92,100). The

decreasing trend over time may be due to the in vivo environment suitability for

islet survival thus decreasing the intrinsic triggering of an apoptotic pathway by

the islet cells. In fact, it appears that islets recovered at 14 days

posttransplantation have apoptotic values similar to those of unencapsulated rat

islets indicating that the islets have recovered from the trauma caused by

encapsulation. In addition, it is likely that the hyperglycemia seen in the

experimental mice may in hibit apoptosis, in P cells, by activating apoptotic

suppresser proteins (31). It is also possible that, in vivo, the microcapsule itself

can contribute to a decrease in islet apoptosis. It is probable that the

microcapsule decreases contact of extracellular apoptotic activators with the

islets (e-g. Fas receptor on the cytotoxic-T-cells contacting its ligand on cell

surfaces) .

Unlike the recovered islets, the in vitro cultured islets exhibited a greater

proportion of apoptotic nuclei as compared to encapsulated islets one day in

culture (Figure 15a). The increase in apoptotic nuclei in the cultured islets

suggests that the in vitro culture conditions promote apoptosis. Media containing

sub-basal glucose concentration (2.8mM) may be the attributing factor for the

increase in apoptotic nuclei in cultured islets. It is thought that low glucose

concentrations can increase p cell apoptosis (31,114,115) by suppressing

inhibition of anti-apoptotic proteins and thus contributing to a higher proportion of

apoptotic nuciei. As previously mentioned, the decrease in insulin stored is

correlated with decreased P cell volume. Decreased insulin stored in the in vitro

cultured islets over time correlates with decreased P cell volume. Decreased P

cell volume further supports the notion of increased P cell apoptosis.

Other studies on the early fate of pancreatic islets were reported by

Davalli et a1 (92,100). However, there are major differences in the experimenial

design of those studies compared to this study. The investigators used

syngeneic unencapsulated mouse islets, and instead of transplanting the islets

into the intraperitoneal cavity of diabetic mice, the islets were allotransplanted

under the kidney capsule. The authors were researching the dynamic changes

that occur in the syngeneic islets transplanted under the kidney capsule in the

immediate posttransplantation period. It was reported that until angiogenesis

occurs (7-10 days), the damage found in the islets was due to cell death. Cell

death was thought to be caused by insufficient oxygen and nutrient diffusion to

the islets inside the kidney capsule. Insulin content as well as P cell apoptosis

were determined in the studies. It was reported that there was a decrease in

insulin content in the islets over time, however the decrease was not significant.

p cell apoptosis was maximal at day 3 and minimal at day 14 posttransplantation.

These researchers also revealed ultrastructural analysis of the islets, using

electron microscopy, to provide evidence for apoptosis and necrosis. Their

findings are quite similar to those of this study in that insulin content decreases

over time in the islets and that apoptosis also decreases the longer the islets

were in vivo. Unlike the kidney capsule transplantation, intraperitoneal

transplantation of microencapsulated islets is less invasive to the recipient. This

issue is relevant when considering that multiple transplantations might be

required to treat a type 1 diabetic. In addition, multiple surgical exposures of the

kidney may cause renal damage. Encapsulated islets are not dependant upon

angiogenesis for nutrients in the immediate posttransplantation period and thus

have adequate access to oxygen and nutrients in that time. Considering the

above mentioned factors, intraperitoneal transplantation of microencapsulated

islets is a favourable method for treating type 1 diabetes.

CHAPTER 6

6. CONCLUSION

This study demonstrates that encapsulated rat islets were functional

throughout the entire in vivo and in vitro observation periods of 14 days. There

was a decrease in insulin content and insulin secretion, over time, in both the

recovered and cultured islets. As compared to the cultured islets, there was a

decrease in the amount of apoptosis occurring in the recovered islets during the

14-day observation period. The level of apoptosis at day 14 posttransplantation

in the retrieved islets had decreased to pre-encapsulation values indicating that

the in vivo environment was more favourable than in vitro environment for islet

survival. If there was any cell death in the islets during the in vivo observation

period, it was likely not due to apoptosis.

Further research needs to be undertaken to determine the fate of

transplanted encapsulated islets in long t e n in vivo studies. With a more

complete understanding of the fate of encapsulated islets and the beginning of

human clinical trials, the number and frequency of transplantations human

patients will require to reverse diabetes will thus be easier to evaluate.

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