Intracranial Transplantation of Pancreatic Islets Attenuates Cognitive and Peripheral Metabolic Dysfunctions in a Rat Model of Sporadic Alzheimer’s Disease

Abstract

Background:

Alzheimer’s disease (AD) is often associated with brain insulin resistance and peripheral metabolic dysfunctions. Recently, we developed a model of sporadic AD associated with obesity-related peripheral metabolic abnormalities in Lewis rats using intracerebroventricular administration of streptozotocin (icv-STZ).

Objective:

We aimed to assess the effect of intracranially grafted pancreatic islets on cognitive and peripheral metabolic dysfunctions in the icv-STZ Lewis rats.

Methods:

AD-like dementia associated with obesity was induced in inbred Lewis rats using a single icv-STZ. Two months after icv-STZ, syngeneic islets (100 islets per recipient) were implanted in the cranial subarachnoid cavity of icv-STZ rats. Morris water maze and marble burying tests were used for studying cognitive and behavioral functions. Central and peripheral metabolic alterations were assessed by histological and biochemical assays.

Results:

The icv-STZ induced increases in food intake, body weight, and blood levels of insulin and leptin without alteration of glucose homeostasis. Grafted islets reduced body weight gain, food consumption, peripheral insulin resistance, and hyperleptinemia. Biochemical and histological analysis of the brain revealed viable grafted islets expressing insulin and glucagon. The grafted islets did not affect expression of brain insulin receptors and peripheral glucose homeostasis. Two months after islet transplantation, cognitive and behavioral functioning in transplanted rats were significantly better than the sham-operated icv-STZ rats. No significant differences in the locomotor activity between transplanted and non-transplanted icv-STZ rats were found.

Conclusions:

Intracranial islet transplantation attenuates cognitive decline and peripheral metabolic dysfunctions providing a novel therapeutic approach for sporadic AD associated with peripheral metabolic dysfunctions.

Keywords

Alzheimer’s disease animal models dementia obesity pancreatic islet transplantation

INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive dementia often associated with peripheral metabolic dysfunctions [1, 2]. There is increasing evidence supporting a link between AD-dementia and impaired insulin signaling in the peripheral tissues and in the central nervous system [3]. Obesity-related peripheral metabolic abnormalities are closely associated with cognitive decline in elderly patients. Peripheral insulin resistance and impaired insulin transport across the blood-brain barrier (BBB) are the most likely causes of obesity-related cognitive impairments [4]. Both insulin-dependent (type 1) and non-insulin dependent (type 2) diabetes have been identified as risk factors for cognitive dysfunctions [5, 6]. Recently, the term “Type 3 diabetes” has been proposed for sporadic AD as a form of “brain diabetes” characterized by elements of both brain insulin resistance and insulin deficiency [7]. Growing evidence supports the concept that impaired brain insulin signaling contributes to the pathogenesis of AD [3 , 9]. In this context, insulin delivery to the brain may provide an efficient tool for correction of AD-associated impaired cognitive functioning. Different routes for insulin delivery to the brain including intracerebroventricular (icv) and intravenous injections were tested during the last decades [10]. In most cases, the insulin delivery to the brain resulted in attenuation of cognitive decline, but invasiveness and risk of hypoglycemia made these routes clinically unacceptable. Currently, the only clinically acceptable method for insulin delivery to the brain of patients is intranasal administration [11]. Several clinical studies in patients with a broad range of cognitive and mood disorders have demonstrated the beneficial effects of intranasal insulin delivery [12, 13] as well as the lack of side effects [14, 15]. The main limitation of the intranasal route is a poor insulin delivery to the central nervous system (CNS). Only a short-term and slightly increased level of intranasally administered insulin in the cerebrospinal fluid (CSF) of healthy volunteers [16] or even a lack of such effect [17] were found.

In order to achieve efficient and metabolically-regulated insulin delivery to the brain, we used a small number of pancreatic islets grafted into the cranial subarachnoid space. Our earlier reports demonstrated that pancreatic islets grafted in the cranial subarachnoid cavity preserved glucose regulated insulin secretion for a long-term period in diabetic rats [18], increased insulin content in the brain, and attenuated behavioral dysfunctions in rats with schizophrenia-like disorder [19, 20]. Recently, we showed that icv administration of streptozotocin (icv-STZ) to Lewis rats induced sporadic AD-like dementia associated with obesity-related peripheral metabolic dysfunctions [21]. In the present report we assessed the therapeutic effects of intracranial islet transplantation on cognitive and peripheral metabolic dysfunctions in Lewis rats exposed to icv-STZ.

MATERIALS AND METHODS

Animals, experimental groups, and study design

10-12-week-old male Lewis (LEW/SsNHsd) inbred rats (Envigo RMS Ltd, Rehovot, Israel) weighing 273 ± 16 g (mean ± SD) were used in all experiments. The experiments were approved by the Tel Aviv University Animal Care and Use Committee. The rats were maintained on a regular 12-h dark/light cycle with access to food and water ad libitum. Standard chow diet was from Envigo RMS Ltd. The animals were divided into three groups, as follows: 1) intact rats; 2) rats exposed to icv administration of streptozotocin (icv-STZ) and two months later to administration of a vehicle (Hank’s Balanced Salt Solution, HBSS) only (STZ-vehicle rats); and 3) rats exposed to icv-STZ and two months later to transplantation of pancreatic islets suspended in the vehicle (STZ-islets rats). Descriptions of biological testing, time points and number of animals per group are presented in Materials and Methods section and in the legends to Figures. An overview of the study design is illustrated in Fig. 1.

Fig. 1

Study design. icv-STZ, intracerebroventricular administration of streptozotocin; Islet Tx, islet transplantation; MB, marble burying test; MWM, Morris water maze test.

Icv administration

Icv injections of artificial CSF (aCSF) and STZ were carried out as described previously [21]. Briefly, 8μl of aCSF or 8μl STZ (3 mg/kg body weight) dissolved in aCSF were bilaterally injected (4μl/2 min in each side) in anesthetized rats using a stereotactic apparatus according to the following coordinates: 0.9 mm posterior, 1.8 mm lateral and 3.8 mm ventral from the bregma level.

Pancreatic islet isolation and transplantation

Islets from the LEW/SsNHsd rats were isolated by enzymatic digestion with a collagenase solution containing 12 units/ml collagenase NB8 and 1 mg/ml bovine DNAse for 15 min at 37°C and purified using a discontinuous histopaque gradient density, as described previously [19]. The purified islets were cultured in CMRL:RPMI medium 1640 (1:1) supplemented with 10% FCS and 1% antibiotics overnight in a CO₂ incubator (5% CO₂ / 95% air) before being transplanted. Islet quantity was expressed as the number of IEQ (islet equivalent), which is calculated based on the number and diameter of the dithizone (DTZ)-stained islets [20].

Islet transplantation procedure was performed as described previously [19] with minor modifications. Briefly, a hole of 1 mm diameter was made in the parietal bone with coordinates: 2 mm to the right and 2 mm posterior to the bregma. The dura mater was carefully incised under stereo-microscope to avoid bleeding from the blood vessels. The hole was used for islet transplantation into the cranial subarachnoid cavity. A polyethylene cannula (1 mm in diameter) containing about 100 IEQ suspended in 15μl of vehicle was inserted into the subarachnoid space. The islet suspension was injected into the subarachnoid cavity by air pressure, the hole was sealed using bone wax, and the incision was closed with surgical suture and disinfected with povidone iodine.

Follow-up of peripheral metabolism

Body weight of rats was recorded during four months after the initiation of the experiment. Food consumption was calculated by deducting the amount of food left by the feeder from the initial recorded amount. The amount of water consumption was determined by subtracting the monitored bottle weights from their initial weights. The measurements were recorded between 9 a.m. and 10 a.m. The average of the food and water consumed per 24 h was calculated for each rat.

Non-fasting blood glucose levels were measured with a portable glucometer (Accu-Check; Hoffmann La Roche, Basel, Switzerland) in whole blood samples. An intraperitoneal glucose tolerance test (IPGTT) was performed in all experimental groups at the end of experiment. A glucose solution (1 g glucose/kg) was injected intraperitoneally after 6 h fasting. Blood glucose was measured before and at 15, 30, 60, and 120 min after glucose injection. Serum levels of insulin and leptin were measured two and four months after beginning of the experiment using ELISA kits for rats (Mercodia AB, Uppsala, Sweden and R&D system, Minneapolis, USA, respectively). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as fasting serum insulin (in mU/L)×fasting blood glucose (mmol/L)/22.5.

Morris water maze (MWM) test

Two months after islet transplantation, spatial learning and memory were tested using MWM. The MWM test was carried out, as described previously [21]. A water maze circular pool (diameter –1.8 m; height –60 cm) filled with water (21 ± 1°C) was used in all experiments. A circular platform (10 cm diameter) was placed 1 cm under the water surface. Rats performed four trials per day (up to 120 s for each trial) on three consecutive days (acquisition phase). On the 4th and 5th days the platform was moved to another quarter of the maze (the reversal phase). The time taken to reach the platform was recorded. If a rat did not find the platform within 120 s, it was manually placed on it for 20 s. In addition, motility parameters were analyzed. Data were recorded using an automated tracking system (Ethovision 3.1 Noldus Information Technology B.V., Wageningen, The Netherlands).

Marble burying test

Marble burying test is based on the observation that rodents bury either harmful or harmless objects (e.g., glass marbles) in their bedding. This test is commonly used to estimate a defensive burying in rodents [22]. Marble burying test was carried out 5weeks after islet transplantation. The rat was placed in a housing cage with 5 cm bedding depth. 20 glass marbles (20 mm in diameter) per cage were placed in 5×4 grid on the bedding. The number of marbles buried during a 20 min period was analyzed. In this procedure, a marble was considered buried if at least 2/3 of the marble was covered with bedding.

Histological analysis of pancreas and brain

Whole rat fixation procedure was carried out using 4% paraformaldehyde perfused via the vascular system. Pancreas and brain were excised, processed for paraffin sectioning (4μm thick) and stained with hematoxylin-eosin (H&E). In addition, pancreas was stained with Masson’s Trichrome. Immunohistochemical analysis (IHC) of pancreas and brain was performed using immunofluorescent and light microscopy. Briefly, histological sections were incubated overnight at 4°C with polyclonal guinea pig anti-insulin antibodies diluted 1:200 (Cell Marque, Rocklin, USA), monoclonal mouse anti-glucagon antibody diluted 1:2000 (Sigma-Aldrich, St Louis, MO, USA), monoclonal mouse anti-α-SMA antibody diluted 1:150 (Cell Marque, Rocklin, USA) and polyclonal rabbit anti-Glut2 antibodies diluted 1:150 (Novus Biologicals, Littleton CO, USA). After washing, the sections were incubated with secondary antibodies diluted 1:400 (Cy^tm3- or Cy^tm2-conjugated affinity pure anti-guinea pig Ig and Cy^tm2-anti-mouse Ig (Jackson Immuno Research Laboratories Inc., Baltimore, PA, USA) for 1 h at room temperature. Nuclei were counterstained with DAPI diluted 1:200. The microscopic images were captured by a digital camera connected to a fluorescent microscope (BX-52, Olympus Ltd, Japan). In addition, the mouse/rabbit PolyScan™ HRP/DAB System (Cell Marque, Rocklin, USA) was used for horseradish peroxidase-labelled detection of insulin, glucagon and Glut2, according to the manufacturer’s protocol. Two–three rats were analyzed per experimental group. Immunostaining of all tested antigens was negative when the primary antibodies were replaced with antibody diluent or normal serum.

Estimation of total islet area and ratio of beta cell to alpha cell areas was made using fluorescent microscopy (BX-52, Olympus Ltd, Japan). Images were captured by digital camera and analyzed by ImageJ Fiji program. The areas occupied by alpha and beta cells were calculated as a percentage of total islet area. At least 80 intrapancreatic islets from each group were analyzed.

Biochemical analysis of the brain

Following euthanasia by CO₂, the brains were removed and the right cerebral hemispheres were frozen in liquid nitrogen and stored at –80°C. 4 ml of cold lysis buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triron X-100, 0.5% sodium deoxycholate) supplemented with protease inhibitors (P2714, Sigma) were added to the whole right brain hemisphere and homogenized using an electric motor grinder (Polytron) on ice for 20 s. To clarify the homogenate, extracts were centrifuged at 12,000×g for 20 min at 4°C (Thermo Scientific Megafuge 16R), the supernatant was removed for determining the protein concentration using a BCA kit (Pierce, Rockford, IL, USA).

ELISA kits were used for measurements of rat insulin (Mercodia AB, Uppsala, Sweden) in the brain extracts according to the manufacturer’s protocols. Glut2 and insulin receptor (IR) expression in the brain was detected by western blot analysis using rabbit anti-Glut2 polyclonal antibody (Chemicon, Temecula, CA, USA) and rabbit anti-insulin receptor polyclonal antibody (Novus Biologicals, Littleton, CO, USA). Equal amounts of protein (50μg protein from each sample) were loaded onto a sodium dodecyl sulphate polyacrylamide gel, using 6% polyacrylamide gels for IR detection, and 10% polyacrylamide gels for Glut2 detection. Separated proteins were transferred onto a nitrocellulose membrane (Whatman GmbH, Dassel, Germany). The membranes were incubated for 1 h at room temperature (RT) in blocking buffer containing 5% skim milk powder dissolved in Tris-buffered saline with 0.01% tween 20 (TBST). The primary antibodies (1:500 in blocking buffer) were incubated overnight at 4°C, and anti-β-actin (1:1000 in Blocking Buffer) were incubated for 2 h at RT with constant shaking. After incubation, membranes were washed with TBST and incubated with anti-rabbit or anti-mouse IgG antibodies (1:3000) for 50 min at RT on a shaker. The bands were visualized using LI-COR Odyssey Imaging system (LI-COR Biosciences, Lincoln NE, USA), and analyzed with Image Studio Lite software (LI-COR Biosciences, Lincoln NE, USA).

Statistical analysis

Data were analyzed using the Statistical Package for the Social Sciences (SPSS) 17.0 software (SPSS Inc., Chicago, IL). Comparisons between the experimental groups for body weight and glucose tolerance were performed using two-way repeated measure analysis of variance (RM-ANOVA) and the Bonferroni post hoc test. Alterations in other biochemical parameters and in the marble burying test were analyzed by one way analysis of variance (ANOVA) and Tukey’s post hoc test. The results were expressed as average ± SE and considered significant at p < 0.05. Cognitive functions in the MWM experiments (escape latency) were evaluated using a Multivariable Cox regression time to event approach in SAS® software, as described previously [21]. The event was defined as finding the platform and the time to event was defined as the time in seconds from placing the animal in the tank to the animal finding the platform. This approach allowed us to investigate the interactions between different treatments and the days of the test. The acquisition phase and the reversal phase were analyzed separately. The MWM motility parameters were analyzed using two ways RM-ANOVA and the Bonferroni post hoc test.

RESULTS

Weight changes and food/water intake

Two-way RM-ANOVA analysis for evaluation of differences in percentage of body weight changes after the icv-STZ, showed significant interactions (F_(7,210) = 6.198; p = 0.0057), therefore we used T-Test for each body weight measurement. Data presented in Fig. 2A show significant body weight decline during the first two weeks after icv-STZ, followed by accelerated weight gain until complete recovery two months after icv-STZ (Supplementary Table 1). The food consumption of icv-STZ rats were significantly (T₍₁₅₎ = 3.095, p = 0.0074) higher than in intact rats (Fig. 2B). At this point, the icv-STZ rats were divided in two groups for future icv-administrations: STZ-vehicle and STZ-islets rats (see Materials and methods section).

Fig. 2

Changes of body weight and food consumption before (A, B) and after islet transplantation (C, D). A) Body weight changes (percentage of initial body weight at day 0) in intact rats (n = 7) and icv-STZ rats (n = 25). *p < 0.01. ^#p < 0.05. B) Food consumption in intact (n = 6) and icv-STZ rats (n = 11). *p < 0.01. C) Food consumption in intact (n = 14), STZ-vehicle rats (n = 5) and STZ-islets rats (n = 6). *p < 0.05. **p < 0.01. D) Body weight changes (gram of difference between day 0 and each time point) in intact rats (n = 9), STZ-vehicle rats (n = 10) and STZ-islets rats (n = 13). *p < 0.05, intact versus STZ-vehicle. ^∧p < 0.05, intact versus STZ-islets. ^#p < 0.05, STZ-islets versus STZ-vehicle. Data presented as average ± SE.

Two months after islet transplantation, we found significant reduction in food consumption of the STZ-islets rats compared to the STZ – vehicle rats (Fig. 2C; one-way ANOVA: F_(2,16) = 13.23, p = 0.0004; Tukey’s post hoc p = 0.0057). Since two-way RM-ANOVA for body weight gain after islet transplantation found significant interaction between time and groups (F_(26,377) = 21.25, p < 0.0001), we performed one-way ANOVA for each time point. One-way ANOVA tests found significant reduction in body weight gain of the STZ-islets rats compared to the STZ-vehicle rats (Supplementary Table 2), although at the end of the experiment the body weight of transplanted rats was significantly higher than in the intact rats (Fig. 2D). No statistically significant differences in water intake were found between all groups at two and four months after beginning of the experiments (data not shown).

Blood glucose and hormones

Neither icv-STZ nor islet transplantation in the subarachnoid space affected non-fasting blood glucose levels. During all the experimental period the physiologically normal range of glycaemia (90–120 mg/dl) was found in rats from all groups. At the end of experiment, intact IPTTG was found in rats from all tested groups (Fig. 3A). Since two-way RM-ANOVA for evaluation of differences in the IPGTT between groups found significant interaction between time and groups (F_(8,148) = 3.913, p = 0.0003), we performed one-way ANOVA for each time point. One-way ANOVA tests found statistically significantly decreased level of glucose (15 min after glucose administration) in STZ- islets rats compared to intact rats (Supplementary Table 3). One-way ANOVA was used to check for differences in peripheral metabolism between the groups. At the end of experiment, peripheral insulin resistance, the levels of insulin and leptin in the blood were significantly increased compared to intact rats. Islet transplantation statistically significantly reduced hyperinsulinemia, peripheral insulin resistance and hyperleptinemia in the STZ-islets rats compared to the STZ-vehicle rats (Fig. 3B-D, Supplementary Table 4).

Fig. 3

Peripheral metabolic changes. A) IPGTT. Blood glucose was measured before and after glucose injection in intact rats (n = 16), STZ-vehicle rats (n = 10) and STZ-islets rats (n = 9). *p < 0.05, intact versus STZ-islets. B) HOMA-IR and C) Blood level of insulin (n = 7–8). *p < 0.05. D) Blood level of leptin (n = 7–8). *p < 0.05, **p < 0.01, ***p < 0.001. Data presented as average ± SE.

Microscopic examination of isolated, grafted, and endogenous islets

Intrapancreatic donor islets showed intensive in vivo DTZ-staining (Fig. 4A). The size range of isolated donor islets was from 50μm to 250μm in diameter, they displayed intact vital morphology (Fig. 4B) and DTZ-staining of zinc–insulin granules (Fig. 4C). At the end of the experiment, morphology and immunohistochemistry of grafted islets (STZ-islets rats) and endogenous islets in intact rats were compared. Figure 5A and Fig. 5B show intact morphology and intensive insulin staining of grafted islets located directly on the pia mater in the cortical subarachnoid cavity. Intact morphology and insulin staining were also found in intrapancreatic islets of intact rats (Fig. 5C, D). Immunofluorescent double staining revealed typical for rodents localization of insulin-producing beta cells in the central area of islets and peripheral localization of glucagon-producing alpha cells in both grafted islets (Fig. 5E) and endogenous islets (Fig. 5F). Double staining for insulin and α-SMA showed localization of maturated blood vessels in close proximity to grafted (Fig. 5G) and to endogenous islets (Fig. 5H). Glut2 localization was primarily detected in the plasma membrane of the islet cells of the grafted (Fig. 5I) and the endogenous islets with low level of expression in intracellular compartments (Fig. 5J). H&E and IHC analysis did not reveal islet-like structures in the cerebral subarachnoid cavity of non-transplanted animals.

Fig. 4

Stereomicroscopic images of donor islets. A) DTZ-stained intrapancreatic islets of donor rats. B) Isolated donor islets. C) Isolated donor DTZ-stained islets. Bars - 300μm.

Fig. 5

Histological images of grafted and endogenous islets. H&E staining (A) and immunoperoxidase insulin staining (B) of grafted islets. H&E staining (C) and immunoperoxidase insulin staining (D) of endogenous islets. Immunofluorescent insulin (red) and glucagon (green) staining of grafted islets (E) and endogenous islets (F). Immunofluorescent insulin (red) and α-SMA (green) staining of grafted islets (G) and endogenous islets (H). Immunoperoxidase Glut2 staining of grafted (I) and endogenous islets (J). Bars: 200μm (A, C-F, H), 50μm (B, G, I, J).

Morphological changes in intrapancreatic islets following icv treatments

At the end of the experiment, the area of intrapancreatic islets in the STZ-vehicle rats (26935 ± 1749μm²) was significantly higher than in the intact and the STZ-islets rats (13235 ± 1675μm² and 18691 ± 1646μm²; F_(2,248) = 16.150, p < 0.0001). At the same time, the ratio of beta cells to alpha cells was slightly decreased in the STZ-islets rats (6.027 ± 0.408) compared to the intact and the STZ-vehicle groups (7.512 ± 0.415 and 7.304 ± 0.433; F_(2,248) = 3.810, p = 0.023). In contrast to STZ-islets rats, the majority of islets in the group of STZ-vehicle rats displayed irregular outlines, localization of alpha cells in the central area of the islets (Fig. 6A, C) and fibrous tissue deposition (Fig. 6B, D). The graphical presentations of quantitative data on islet’s area and the ratio of beta cells to alpha cells are shown in Fig. 6E, F.

Fig. 6

Histological images of intrapancreatic islets. Immunofluorescent insulin (red) and glucagon (green) staining (A, C) and Masson trichrome staining (B, D) of STZ-vehicle and STZ-islets rats. Bars: 100μm. The graphical presentations of quantitative data on islet’s area (E) and ratio of beta cells to alpha cells (F). *p < 0.05. **p < 0.0001.

Expression of insulin, IR, and Glut2 in the brain

The extracts prepared from the whole right cerebral hemispheres were used for insulin measurement by Elisa. One-way ANOVA test for insulin levels in the brain found statistically significant elevation of insulin content in the brain of STZ-islets rats (F_(2,14) = 8.804, p = 0.0033; Fig. 7A) compared to non-transplanted animals (STZ-islets versus intact p = 0.0064; STZ-vehicle versus STZ-islets p = 0.0090). IR and Glut2 expressions in the whole right hemispheres were analyzed using western blot. One-way ANOVA did not find statistically significant differences in IR and Glut2 expression between all tested groups (F_(2,15) = 0.6563, p = 0.533; Fig. 7B) and (F_(2,16) = 2.439, p = 0.1189; Fig. 7C), respectively. Immunohistochemistry analysis confirmed membrane and cytoplasmic expression of Glut2 in hippocampal (CA3) and hypothalamic (dorsomedial nucleus) regions of the STZ-islets and non-transplanted rats (Fig. 7D-G).

Fig. 7

Insulin, IR, and Glut2 expression in the brain. A) Insulin content measured by ELISA (n = 5–6). *p < 0.001 versus intact and STZ-vehicle groups. Data presented as average ± SE. B) Expression of IR and C) Glut2 detected by western blot analysis (n = 5–6). Quantitative analysis was normalized to the β-actin level. D, E) Immunoperoxidase Glut2 staining in hippocampal (CA3) and (F, G) hypothalamic (dorsomedial nucleus) regions of the STZ-islets (D, F) and STZ-vehicle (E, G) rats. Bars: 50μm.

Morris water maze test

Statistical analysis of the MWM test is shown in Supplementary Table 5 and results graphically presented in Fig. 8A, B. In the acquisition phase of the MWM test, a Multivariable Cox regression time to event approach found that the STZ-vehicle rats demonstrated severe impairment in spatial memory as expressed by a significantly increased latency to the platform compared to both the intact rats and the STZ-islets rats. In contrast to the intact and the transplanted rats, the STZ-vehicle rats manifested a profound deficit in learning capacity. In the reversal phase of the MWM test, the STZ-vehicle rats but not STZ-islets rats showed a trend increase in latency to the platform relative the intact rats on day 4. No statistically significant differences between the STZ-vehicle and the STZ-islets rats were found, whereas both groups showed impairment in spatial memory and learning compared to the intact rats on day 5 of reversal phase (Fig. 8A). Since two-way RM-ANOVA for evaluation of differences in mobility parameter (velocity) between groups found significant interaction between days and groups (F_(4,28) = 7.609, p = 0.0003), we performed one-way ANOVA for each time point. One-way ANOVA tests for differences in mobility parameters (velocity) did not reveal statistically significant differences between the STZ-vehicle and the STZ-islets group in both acquisition and reversal phases (Fig. 8B). However, elevated velocity of the intact rats were found at the first day (F_(2,14) = 9.689, p = 0.0023) compared to the STZ-islets rats (p = 0.0091) and the STZ-vehicle group (p = 0.0079). Contrariwise, reduction in velocity of the intact rats was found at the third day (F_(2,14) = 4.063, p = 0.0406; Tukey’s test was not significant) compared to the STZ treated groups.

Fig. 8

Behavioral functions. MWM task. A) Escape latency. B) Velocity. Intact (n = 8), STZ-vehicle (n = 10) and STZ-islets (n = 13). *p < 0.05 versus STZ-islets and STZ-vehicle rats. ^#p < 0.05 versus STZ-vehicle rats. ^∧0.05 < p < 0.06 versus intact rats. C) Marble burying test (n = 7–8) *p < 0.001 versus intact and STZ-islets rats. Data presented as average ± SE.

Marble burying test

One-way ANOVA test for differences in marble burying test shows statistically significant (F_(2,19) = 11.70, p = 0.0005; Fig. 8C) reduction in number of marbles buried in the STZ-vehicle rats compared to the intact rats (p = 0.0065). This STZ-induced impairment in burying behavior was not detected in the STZ-islet group (p = 0.0005 versus STZ-vehicle).

DISCUSSION

In this preclinical study, we tested the effects of intracranial transplantation of pancreatic islets on cognitive and metabolic abnormalities in rat model of sporadic AD. Early reports on experimental pancreatic islet transplants in the CNS for the treatment of peripheral metabolic dysfunctions were published several decades ago and recently were summarized in a review article [23]. In most cases, intracranial and intrathecal pancreatic islet transplantations were used as a tool for treatment of diabetic animals [18 , 24–26]. Only one study focused on body weight reduction following intraventricular pancreatic islet transplants in intact rats [27]. Taken together, the early reports indicate that immunoprivileged and highly oxygenated CNS transplantation sites provide an optimal microenvironment for survival of grafted islets. Long term metabolically regulated insulin secretion from CNS grafted islets resulted in stable normoglycemia in diabetic animals. Recently, we showed that a marginal islet mass grafted in the cranial subarachnoid cavity provided efficient insulin delivery to the brain and attenuated behavioral and cognitive dysfunctions in rats with schizophrenia-like disorder [19].

The present study was design to evaluate the efficacy of intracranial transplantation of a marginal islet mass for the treatment of cognitive and peripheral metabolic abnormalities in Lewis rats with sporadic AD induced by single icv-injection of STZ. Data presented in our previous report [21] and in the current study demonstrate the development of a stable and severe dementia in the Lewis rats associated with obesity for period of four months after icv-STZ administration. Moreover, our most recent data indicate that severe cognitive decline and obesity-related metabolic dysfunctions in the Lewis rats were preserved after icv-STZ for at least eight months (Bloch K, unpublished data). In the inbred Lewis rat strain, the icv-STZ induced a rapid body weight decline during the first two weeks followed by accelerated weight gain. As a result, the icv-STZ rats attained the body weight range of intact rats of the same age about two months after the initiation of the experiment. At this point, icv-STZ rats expressed intact glucose homeostasis, elevated food consumption, hyperinsulinemia, peripheral insulin resistance, hyperleptinemia and severe cognitive decline, as it was shown previously [21]. This time point, which is characterized by peripheral metabolic abnormalities and cognitive dysfunctions, was chosen for therapeutic intervention in the icv-STZ rats.

In order to determine the effect of grafted pancreatic islets on cognitive and peripheral metabolic dysfunctions, one hundred pancreatic islet equivalents (IEQ) were injected in the subarachnoid cavity of the icv-STZ rats. This marginal mass of grafted islet is about 20–30 times less than the number of transplanted islets required to achieve a stable normoglycemia in diabetic Lewis rats [18, 28]. Two months after islet transplantation, viable islets were found in the cranial subarachnoid cavity. Histological analysis revealed intact islet morphology, intensive expression of both islet hormones (insulin and glucagon) and Glut2 (the major glucose transporter isoform in insulin producing beta-cells). The high level of insulin found in the whole brain of the transplanted animals reflects mainly presence of islet-derived insulin. In our previous reports, we showed that a small number of intracranially transplanted naked or alginate encapsulated islets were able to increase insulin concentration in the whole brain and in the cognitive-relevant areas such as hippocampus and frontal cortex [19, 20]. At the same time, the brains of non-transplanted rats had a barely detectable level of insulin. These data agree with previously published reports on very low brain insulin level [10, 20]. The long-term viability of functional islets in the subarachnoid cavity of Lewis rats was confirmed in our most recent study demonstrating increased brain insulin content and presence of viable pancreatic islet grafts six months after islet transplantation (Bloch K, unpublished data). Importantly, the intracranial pancreatic islet grafts did not affect intact peripheral glucose homeostasis during the whole post-transplantation period as it was shown in this study and in our previous published works [19 –21].

In the present study, we show the beneficial effects of intracranially grafted pancreatic islets on systemic metabolism leading to normalization of food consumption and attenuation of body weight gain in the icv-STZ obese rats. These results are in agreement with an earlier study demonstrating weight loss in intact Lewis rats following intraventricular transplants of syngeneic pancreatic islets [27]. Presumably, reduced food intake and decreased body weight gain after intracranial islet transplantation are at least in part a result of the well-known anorexigenic effect of central insulin activity [29, 30]. In the icv-STZ Lewis rats, the reduction in body weight gain after islet transplantation was accompanied by attenuation of hyperinsulinemia, peripheral insulin resistance, and hyperleptinemia. Such beneficial systemic metabolic changes confirm previous results on metabolic effects of central insulin administration [3, 30]. In the pancreas, icv-STZ induced islet hypertrophy, intra-islet fibrosis, and abnormal islet cytoarchitecture. Such islet abnormalities were previously found in a variety of animal models of obesity [31, 32] and in obese humans [33, 34]. The data presented in this study demonstrate that intracranial pancreatic islet transplantation may provide an efficient tool for correction of obesity-related systemic and pancreatic dysfunctions.

In addition to correction of peripheral metabolic impairments, the intracranial islet transplantation attenuated cognitive and behavioral dysfunctions in the icv-STZ Lewis rats. Spatial learning and memory estimated in the acquisition phase of MWM test were significantly improved after pancreatic islet transplantation compared to the non-transplanted icv-STZ rats. Importantly, the transplanted and non-transplanted rats with AD did not differ statistically in the speed of their swim. In addition, the differences in MWM performance on second and third days between intact rats and obese STZ-treated animals cannot be explained by fluctuations in velocity. Thus, the main differences in the MWM test performance reflect changes in cognitive functions, rather than in locomotor activity. Noteworthy, in the reversal phase of MWM test, the long-term retention of spatial memory in the group of transplanted rats was only slightly better compared to non-transplanted obese rats. Recently, amelioration of cognitive dysfunctions studied in the acquisition phase of the MWM test was also found in icv-STZ Sprague–Dawley rats after long-term treatment with intranasal insulin, suggesting an important role of brain insulin in regulation of learning and memory capacities [35]. The marble burying test was carried out to assess the effect of intracranial pancreatic islet transplantation on defensive digging behavior in rats exposed to icv-STZ. Previously reported results showed that transgenic AD mice (Tg-APP/PS1) buried fewer marbles than non-transgenic mice [36]. In the present study, we also show that icv-STZ leads to decreased digging, while grafted islets completely restored burying behavior. It can be speculated that the beneficial effect of grafted islets reflects attenuation of a putative hippocampal dysfunction that may be involved in the aberrant digging behavior [37]. The important role of hippocampal dysfunction in development of sporadic AD in icv-STZ rats was discussed in recent review articles [38, 39].

We have previously shown that intracranial pancreatic islet transplantation increased insulin content in the hippocampus, the hypothalamus [19], and in the whole brain of Lewis rats [20]. In this study, we focused on a possible effect of the grafted islets on the levels of cerebral IR and Glut2 at the late stage of the icv-STZ model. Both IR and Glut2 play important role in brain glucose metabolism. Moreover, Glut2 is required for efficient STZ cytotoxicity [40]. These two proteins were suggested to be acute targets of streptozotocin in icv-STZ rats [41]. Since IR and Glut2 are broadly expressed in various areas of the brain [42 –44], the levels of these proteins were measured in whole brain extracts using western blot analysis. About two-fold decrease in the level of Glut2 expression was found in rats exposed to icv-STZ, but the differences between the groups did not reach the conventional significance level (p < 0.05) and therefore reflect a statistical trend only. The trend to reduction in expression of Glut2 protein in icv-STZ rats was not seen in islets-transplanted rats. These differences may reflect a possible involvement of brain endogenous Glut2 in the attenuation of cognitive dysfunctions in the icv-STZ rats following islet transplantation. However, the direct contribution of grafted islets expressing Glut2 cannot be disregarded. In contrast to other reports, we did not detect significant alterations in the level of cerebral IR between the intact and the icv-STZ rats. The grafted islets also did not affect the IR expression in the brain. Yet, it is possible that one or several post-IR alterations may be involved in impaired insulin signaling in the icv-STZ Lewis rats. Furthermore, direct involvement of brain IR expression in the development of behavioral dysfunctions was shown recently [45] and thus cannot be ultimately excluded. Additional research is needed to clarify the involvement of Glut2 and IR in pathogenesis of sporadic AD and their role as potential targets for therapeutic interventions.

In conclusion, transplantation of a marginal number of pancreatic islets into the cranial subarachnoid cavity attenuates cognitive decline and peripheral metabolic dysfunctions in the Lewis rats with icv-STZ-induced AD-like dementia. Such type of cell therapy provides a novel and promising treatment of sporadic AD associated with obesity-related metabolic abnormalities.

Footnotes

ACKNOWLEDGMENTS

The authors thank Dr. Daniel Lazard for fruitful discussions and critical reading of this paper and Prof. Daniel Offen for providing a stereotactic apparatus. This study was supported in part by grants (0601243032 and 0601523471) from Tel Aviv University (KB) and Mayer Foundation for Research (AW).

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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