Stroke-induced stem cells proliferation in normal versus diabetic mice and pharmacological regulation

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STROKE-INDUCED STEM CELLS

PROLIFERATION IN NORMAL VERSUS DIABETIC MICE AND

PHARMACOLOGICAL REGULATION

Stroke-inducerad stamcells proliferation i normala kontra diabetiska möss och famakologisk reglering

Zainab Fadhel

Faculty o f Health, Natural and Engineering Sciences Department o f Chemical Engineering

Chemical Engineering 30 hp

Supervisor: Cesare Patro ne (Södersjukhuset), Vladimer Darsalia (Södersjukhuset), Eewa Nånberg (KAU) Examiner: Lars Järnströ m

Date:15 -05-25

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Abstract (swedish)

Bakgrund: Stroke är en av de primära orsakerna till mortalitet i västvärlden och typ 2 diabetes är en riskfaktor tätt kopplat till stroke. Stroke leder till en märkbar ökning av proliferande neurala stamceller i den subventrikulära zonen i den drabbade hjärnhalvan, och ökar neurogenes hos både vuxna gnagare och i human hjärna, vilket tros leda till en viss

återhämtning efter stroke. Neurogenes hos typ 2-diabetes patienter är försämrad. Exendin-4 är ett läkemedel för klinisk behandling av typ 2-diabetes som har visat neuroprotektiva

egenskaper i djurförsök. Det är dock oprövat huruvida behandling med Exendin-4 ger ökad neurogenes efter stroke i kliniskt relevanta situationer.

Syfte: Syftet med denna studie var att undersöka om stroke-inducerad stamcells proliferation påverkas av experimentell diabetes i möss, samt om Exendine-4 reglerar stroke-inducerad stamcells proliferation i normala versus diabetiska möss.

Material och Metoder: Medelålders överviktiga/diabetiska möss fick stroke inducerat.

Behandling med Exendin-4 påbörjades 1,5 timmar senare och fortgick två gånger dagligen i en vecka innan djuren avlivades. Hjärnor från mössen isolerades och färgades med den specifika proliferation markören Ki67. Antalet prolifererande neurala stamceller

kvantifierades sterologiskt genom att räkna Ki67⁺ celler i den subventrikulära zonen i båda (kontroll resp. stroke) hemisfärerna.

Resultat: Antalet prolifererande stamceller var statistiskt signifikant högre i de normala mössen versus diabetiska möss i de båda sidorna av subventrikulära zonen. Behandling med Exendine-4 gav statistiskt signifikant ökad proliferation av stamceller i normala möss men inte i diabetiska möss.

Slutsatser: Resultaten i denna studie visade att typ 2 diabetes gav en försämrad proliferation av neurala stamceller i den subventrikulära zonen, och att Exendin-4 förbättrade

proliferationen i den subventrikulära zonen i en preklinisk modell med klinisk relevans.

Resultaten antyder att Exendin-4 kan ha potential för att administreras i ambulansen eller akutvårdsavdelning till normala patienter med stroke men ytterligare studier krävs.

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Abstract English

Introduction: Stroke is caused from the occlusion of any cerebral artery leading to cerebral ischemia, brain damage and consequent neurological impairments and disability. The primary causes of mortality in western populations is stroke. Diabetes type 2 is a high risk factor for stroke. Stroke leads to an observable increase of neural stem cell proliferation in the subventricular zone and enhances neurogenesis in the adult rodent and human brain which suggest a mechanism contributing to stroke recovery. Neurogenesis in type 2 diabetes patients is impaired. However, whether stroke-induced neurogenesis is impaired in diabetes has not been studied. Exendin-4 is a drug for clinical treatment of type 2 diabetes which has been shown to have neuroprotective properties in animal studies. However whether Exendine-4 leads to increased neurogenesis after stroke in the diabetic brain has not been previously studied.

Aims: The specific aims of this project were to determine whether stroke-induced stem cell proliferation is impacted by diabetes in the mouse, and if Exendine-4 regulates stroke-induced stem cell proliferation in normal and diabetic mice.

Material and Methods: Aged obese/type 2 diabetic mice were subjected to stroke. The Exendin-4 treatment was started 1.5 hours thereafter. Treatment was continued for one week before animals were sacrificed. Brains were isolated and the neurons were immunostained using the specific proliferation marker Ki67. Neural stem cell proliferation was quantified by counting Ki67+ cells in the ipsilateral (subventricular zone in stroke hemisphere).The

estimation was assessed by stereological counts of proliferating stem cell in the subventricular zone.

Results: The number of proliferating stem cell after stroke was statistically significantly higher in the normal mice versus diabetic mice. The effect was present in both sides (control and stroke) of the subventricular zone. Exendine-4 treatment induced statistically significant increased of stem cell proliferation in normal mice but not in diabetic mice.

Conclusions: The result of this study shows that type 2 diabetes decreased the proliferation of neural stem cell in the subventricular zone and that Exendin-4 enhanced the subventricular proliferation in a preclinical model of clinical relevance. The data suggest that the Exendin-4 treatment could be administered to normal patients suffering from stroke in the ambulance or in the emergency room although more studies are needed.

Keywords: Stroke, diabetes, GLP-1, Exendin-4, neurogenesis, animal model, mouse

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ABBREVIATIONS

tPA - Tissue plasminogen activator GLP-1 – Glucagon-like peptide 1

GLP-1R – Glucagon-like peptide 1 receptor Ex-4 – Exendin-4

T1D – Type 1 Diabetes T2D – Type 2 Diabetes HFD – High Fat Diet NSCs - Neural stem cells DAB- diaminobenzidine SGZ- Subgranular zone SVZ- Subventriculer zone CNS - Central nervous system SEM- Standard error of the mean

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1. Introduction

1.1 Stroke

Stroke is major causes of disability and death in many Western countries were the incidence of stroke increases along with the ageing [1]. Ischemic stroke occurs in 85% of all the stroke cases. It is caused from the occlusion of any cerebral artery leading to cerebral ischemia, brain damage and consequent neurological impairments and disability. Various factors such as hypertension, diabetes mellitus, coronary heart disease, obesity, sex and race increases the risk of stroke [2, 3]. Almost 40% of the patients are unable to recover fully from the disabilities that may follow [3].

Thrombotic stroke is characterized by a thrombus obstructing the blood vessel and blocking the blood flow to a specific brain area. A surgical removal of the clot can be efficient in the first 3-4 hours after stroke but most of the patients cannot benefit of this treatment due to late arrival at the hospital, delayed diagnosis or contraindications[4] .

Today, recombinant tissue plasminogen activator (r-tPA) is the only established

pharmacological treatment that restores blood flow to the brain [5]. On the other hand, only a low amount of patients receives r-tPA due to short effective therapeutic window (up to 4.5 h from stroke symptom onset). Also r-TPA has been related with increased risk of intracerebral hemorrahagic transformation and therefore even less treated frequently in diabetic patients with stroke due to their weakened vascular system when compared to healthy individuals, since elevated blood glucose and diabetes is associated with increased risk of intracerebral hemorrhages after r-TPA use. While it is still unclear if glucose lowering strategies can be beneficial against stroke, it is well understood that hypoglycemic events should be avoided after stroke in T2D patients suffering stroke, since neurons critically depend on glucose as a source of energy[16]. Hypoglycemia can be an result from glucose lowering treatments and therefore insulin might not be the greatest drug in that view [5, 6,7]. Moreover,

neuroprotective strategies against stroke failed to be translated clinically along the past decades [8,9]

1.2 Type 2 Diabetes

There are two main forms of diabetes, classified by etiology: type 1 diabetes (T1D) and type 2 diabetes (T2D). The majority of the diabetic patients have T2D, and previously it was a disease of older adults, but T2D is rapidly increasing among young people The main causes of the syndrome appear to be various and include obesity (especially abdominal obesity), physical inactivity, insulin resistance, aging, and a genetic predisposition[10]. T2D is

characterized by insulin resistance leading to progressive decline in functional β-cell mass and impaired insulin production. The mechanism behind this occurrence is not really clear, but it is believed that it is a result of chronic oxidative stress on beta cells due to lipotoxicity and

glucotoxicity[11].

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1.3 Stroke and type-2 diabetes

There are several risk factors associated with stroke and T2D is a major risk for stroke, that arises already in pre-diabetic stages, where insulin resistance is a strong risk factor for stroke [12-15]. Furthermore, T2D and stroke are common causes of morbidity/mortality among middle-aged adult in Europe and North America. Finally, T2D patients suffering from a stroke have an increased risk of stroke recurrence and dire prognosis in terms of survival and functional recovery [13-15].

1.4 Type 2 diabetes and worsened stroke outcome

The pathogenic mechanisms leading to worsened stroke outcome in T2D are not yet clear.

Admission hyperglycemia is a common risk factor for increased brain damage in stroke.

However, there is uncertainty whether glucose lowering treatments after stroke could benefit the clinical outcome[16]. Insulin resistance observed in T2D could have detrimental effects on brain metabolism leading to, among other things, increased oxidative stress and mitochondrial dysfunction [17,19]. These events may lead to neuronal/microvasculature damage, which may predispose the brain to inflammation resulting in further synapse deterioration and heath decline [17,19] Thus, the T2D brain presents a combination of damaging factors that might increase brain damage after stroke. However, these factors need to be precisely identified and their targeting proven to be therapeutically useful.

1.5 Neurogenesis Type 2 diabetes and Stroke

We know today that neural stem cells (NSCs) can generate neurons in the mammalian brain through a process named adult neurogenesis[20]. A few decades ago, the prevailing idea was that all neurons were developed before birth in the human brain and no new neurons were added to the central nervous system (CNS) in the adult life. Neurogenesis has been clearly demonstrated at three locations under normal conditions: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus[20]

and recently studies have shown neurogenesis in the hypotalamus[21]. Neurons born in the adult SVZ migrate over a great distance through the rostral migratory stream and become granule neurons and periglomerular neurons in the olfactory bulb (OB). Recent studies also showed that newborn neurons in the adult brain integrate into the existing circuitry and receive functional contribution.

Moreover, these cells may be necessary for certain forms of brain function involving the olfactory bulb and the hippocampus, which is important for some forms of learning and memory[20]. Stroke leads to an observable increase of cell proliferation in the subventricular zone. Stroke-generated new neurons, as well as neuroblasts migrate into the damaged area in striatum, where they express markers of developing and mature, striatal medium-sized spiny neurons. Thus, stroke enhances neurogenesis in the adult rodent and human brain and this process has been suggested to play a role for a functional recovery[22] suggesting a

contribution of NSCs to stroke recovery. Interestingly, neurogenesis in T2D is impaired[23].

However, whether stroke-induced neurogenesis in T2D is impaired too and/or whether it can

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be pharmacologically stimulated remains to be studied. Stroke induces neurogenesis in both rodents and human and this process has been suggested to play a role for a functional recovery[41].

1.6 Glucagon-like receptor 1 Agonist

Endogenous Glucagon-like receptor 1 (GLP-1) is produced by L-cells in the colon in response to food ingestion [24]. However, GLP-1 is degraded in a matter of few minutes by the

enzyme dipeptidyl peptidase-4(DPP-4) and hence also excluded as a drug for treatment of diabetes. Exendin-4 (Ex-4) is a GLP-1 analog that is isolated from the saliva of the “Gila Monster” lizard, the substance is stable and resistant to degradation(25). It is a 39 amino acids long peptide that shares approximately 53% of the amino acid identity with the native GLP-1 that can be found in the human body[24] (see figure 1 for a comparison in sequence). GLP-1R agonists such as Ex-4 are new treatments for T2D [26] which amplify glucose-dependent insulin secretion but do not cause hypoglycemia[27]. Interestingly, GLP-1R mRNA and protein are highly expressed throughout the brain[28] and GLP-1and Ex-4can cross the blood-brain-barrier[29]. Growing evidence from our and other groups suggests an important role for GLP-1R agonists as neuroprotective effect against stroke sequele[30,31],

Parkinson’s[32] and Alzheimer’s[33,35] and also possessing neurogenic and anti- inflammatory features[32,35]. On the other hand the mechanisms behind the effects are poorly investigated.

Figure.1 . Exendin-4 structure in comparison with GLP-1.

2. Project Hypothesis

Although recent report have shown that adult neurogenesis is impaired by T2D, whether this is the case following stroke has not been previously investigated. We hypothesize that T2D decreases the neurogenic response after stroke thus leading to a decreased plasticity response and recovery. GLP-1R activation has been shown to stimulate adult neurogenesis in non- diabetic rodents. However whether GLP-1R activation leads to increased neurogenesis after stroke in the diabetic brain has not been previously studied. Since studies have shown that

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increased stem cell proliferation also stimulate the neurogenesis, we aim to determine that in this project.

3. Aims

The specific aims of this project were to determine whether:

1) Stroke-induced proliferation of NSC:s is impacted by diabetes in the mouse

2) Ex-4 treatment affects stroke-induced proliferation of NSC:s in normal and diabetic mice

4. Material and Methods

4.1 Experimental Design

This project involved two in vivo experimental studies with two groups of mice in each study.

Study 1: Sixteen 2-month old mice received 5 µg/kg Ex-4 intraperitoneally (i.p.) at 1.5 hours (n = 8) after tMCAO. Control mice (n = 8) were given vehicle injection 1.5 hours after

tMCAO. The treatment continued for 7 days with the dose of 0.2 µg/kg given daily until sacrifice.

Study2: Eighteen 2-month-old mice were exposed to high-fat diet (HFD; see below) for 12 months to induce T2D/obesity (see below). Body weight and blood glucose levels were monitored throughout the experiment. At the end of the diet period, mice were subjected to stroke and received 5 µg/kg Ex-4 i.p. at 1.5 (n = 9) after MCAO. Control mice (n = 9) were given vehicle injection 1.5 hours after MCAO. The treatment continued for 7 days as in Study 1 until sacrifice (see Fig. 2). The mice were sacrificed 1 week thereafter and their brains were extracted and stored in 20% sucrose solution and preserved in a refrigerator at 4° Celsius.

The experimental work in this thesis project started at this time-point where the investigation of how stroke-induced neurogenesis is affected by diabetes and -whether Ex-4 regulates stroke-induced neurogenesis in normal and diabetic mice was investigated.

4.2 Immunocytochemistry for neural stem cells and neuroblasts

The brains were cryosectioned and the proliferating cells were immunostained with the proliferation marker Ki67 ( see below). The appearance of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be absolutely detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G₁, S, G₂, and mitosis), but is absent from resting cells (G₀), makes it an excellent marker for Neural stem cell proliferation. Although the Ki-67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear[36]. The proliferation of stem cell was quantified by counting Ki67+

cells in the ipsilateral stroke hemisphere in the subventricular zone. In order to determine whether Ex-4 modulate neurogenesis after stroke in normal mice in striatum, the specific neuroblast marker doublecortin (DCX) was used to label microtubule cytoskeleton in

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immature neurons. The antibodies incubation, washing and quantification of the cells was performed by me under the close observation of my supervisor. The tMCAO and extraction of the mice brains was performed by a specialized team at Karolinska Institutet in Huddinge.

Figure 2: The study design shows that the mice were given HFD starting from 2 month after birth. HFD was given for 12 month. Stroke was induced by tMCAO when the mice was 14 month and Ex-4/vehicle were given from 1,5 hour after tMCAO. The mice were kept alive for 1 week to evaluate the effects of the drug.

4.3 The HFD T2D Animal model

To induce obesity and T2D the mouse strain C57Bl/6J which develops a condition similar to T2D and obesity when fed a HFD [23] was used. Also, as with human beings with T2D, the C57Bl/6J fed on a HFD goes through different stages of insulin resistance, glucose

intolerance and beta-cell apoptosis during the development of an overtly obese and diabetic state [23]. Starting from the age of 8 weeks the mice were fed a HFD (Research Diets, Inc., New Brunswick, NJ) for 12 month.

4.4 Transient middle cerebral artery occlusion (tMCAO)

The animals were first anesthetized with 3% isoflurane and the anesthesia continued during surgery with 1.5 % isoflurane with a snout mask. The procedure of inducing stroke by tMCAO starts with exposing the carotid arteries on the left side, then the external carotid was ligated and temporary sutures were placed over the common carotid artery. Through a small incision in the external carotid artery, a 7-0 silicone-coated monofilament (0.17 mm in diameter) was advanced through the internal carotid artery to the origin of the MCA to block it, the wound was closed and the animal was allowed to wake up. After 30 minutes of occlusion, the animals were anesthetized again, the filament was withdrawn and the ligatures were removed from the common carotid artery. During surgery, body temperature was maintained at 36-38°C with a heating pad. The mice were transferred in a heated box for 2h and they were allowed to wake up again. The surgeon performing the operation was blinded to the experimental groups

4.5 Immunohistochemistry (IHC)

Animals were deeply anesthetized with an overdose of sodium pentobarbital and perfused transcardially with 4% paraformaldehyde. The brains were extracted, post-fixed in 4%

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paraformaldehyde overnight at 4°C and submersed in 20% sucrose in phosphate buffer (PBS) until they sunk (indicated the saturation of the tissue with sucrose). Using a sliding microtome, slices of 40µm thickness were cut and stained as free-floating sections. The following primary antibodies were used: anti-Ki67 (1:500; Millipore, MA) a proliferation marker to stain proliferating cells in SVZ and rabbit anti-DCX (1:1000 dilution, Wako Chemicals) a neuroblasts marker for microtubule-associated protein expressed by neuronal precursor cells while actively dividing in the striatum.

Sections were incubated with the primary antibody for 24-36h at 4°C in PBS containing 3%

goat serum and 0.25% Triton-X. The bound primary antibody was detected using a biotin- conjugated (Vector, CA) secondary antibody (1:200) against the host species of the primary antibody. The incubation was applied for 2h at room temperature (approx. 18-21ºC) in PBS containing 3% of the goat serum and 0.25% Triton-X. For chromogenic visualization, avidin- biotin complex (ABC kit, Vector, CA) and diaminobenzidine (DAB) were used to assure contrast effect. The thickness of the tissue shrinks after all the staining procedure to about 10- 15 µm. The mounting of sections was made on gelatine coated microscopy glass of 1mm and cover glasses of 0.1mm.

4.6 Cell quantification

The measurements were performed using NewCast (Visiopharm, Hoersholm, Denmark) software. The Ki67- and DCX-stained tissue sections were displayed live on a computer monitor, this being connected to an Olympus BX51 microscope.

The number of proliferating stem cells (Ki67⁺) and neuroblast (DCX) was quantified using the fractionator method explained below. Quantifications were performed using an 40x air objective with numeric aperture driven by NewCast software. The focus plan was moved through the thickness of the tissue so that all the visible cells were counted. Three sections were mounted on the microscopy glass for the counting. Immunoreactive cells were counted using a computerized non-biased setup for stereology, driven by NewCast software.

4.7 The fractionator and nucleator

The brains were cut 40 µm thick consecutive sections. For stereological quantification every 8^th section was used, thus the section fraction was defined as 1/8. Because the sections in each series (containing every 8^th section) represented 1/8^th of the whole brain, the total number of cells in the brain could be mathematically estimated by multiplying the counted number of cells by the section fraction (fractionator principle)[37,38].

The count was performed in subventricular zone and it included three section of every 8th brain section, see appendix for number of samples from each study .In Fig. 3A, using the software, a dashed line was used to mark these regions and the area around the contour and inside the delimitation were included in the counting. After this delimitation, an evenly spaced grid (see the black squares in Fig. 3A was applied with the aid of the software were

parameters like step length and space outside the contour to be excluded were set. The step length represents the space between the black grids from figure 3, and determines in how many samples (or frames) the counting will be performed. The step length is chosen

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according to area that is selected, which in this case is represented by the whole area of SVZ.

The counting is done manually and the data was saved in an excel file in the software. The grid was applied 100% over the SVZ. The direction of the grid was randomly chosen by the software. The next step is to count in every square of the grid with a 3 colored counting frame, like in Figure 3B . This frame systematically moves from one square to another, which means the frame will have the same position in every square. The frame has a red and a green line. The cells being inside the frame but crossing the red line are not counted, all the rest being added to the quantified number (see Figure 3B).

The total number of cell can be estimated by using the following formula:

𝑁𝑁 = �𝑄𝑄 × 1 𝑠𝑠𝑠𝑠 ×

1 𝑎𝑎𝑠𝑠

N represents the estimation of the total number of cells. Q is the number of sampled cells using the method described above, (sf) is the section fraction and is the 1/8^thof the whole brain. Area fraction (af) is calculated by dividing the area of the counting frame by the square distance between each grid (the black squares in Figure 3A) in order to cover the entire surface of the counting area that was performed.

Figure 3. Stereology used to quantify cells after IHC of Ki67+ cells A. Dashed green lines delimitates the area of the subventricular zone. The black squares

represents grids equally distanced over subventricular zone where the counting is performed.

The pictures were acquired with the NewCast software and x10 lens. B. Frame used to count inside every grid from (A). The cells intersecting the green line are counted (+) and the ones intersecting the red line are not counted (-).The pictures were acquired with the NewCast software and x40 lens

4.8 Data Analysis/Statistics

Extracted data was analyzed using prism 6.0a (Graph Pad Inc.). Statistical analyses for Ki67 was performed by using the one-way analysis of variance ( ANOVA ) between the four groups followed by Tukey's multiple comparisons test, Which is a common precise method to see if significant differences existed between the test groups when comparing the mean values

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of the number of Ki67+ cells. Differences between the groups were considered statistically significant when P< 0.05. Data are presented as means +/- SEM ( Standard error of the mean), quantifies how precisely we know the true mean of the samples. It takes into account both the value of the SD (standard deviation) and the sample size. For the DCX analysis the unpaired t-test was used between the vehicle treated and Ex-4 treated normal mice.

4.9 Ethical Considerations

To study stroke we have to unfortunately sacrifice animals, there are no validated models for in vitro studies. Animal models that mimic human stroke are MCAO model that is employed in this study. The positive results that many drugs have proven in animals, has negative results in humans. Which is mainly due to the drugs tested too shortly according to the time when stroke is induced which gives irrelevant results clinically. We tried to create preclinical data that provides clinical relevance, by emulating the real situation stroke patients often find themselves in. Thus initiated Ex-4 treatment 1.5 hours after stroke. Which also eventually reduce the number of animals needed to study the efficacy of Ex-4.In order to avoid

unnecessary suffering, we keep the animals in good environment, avoiding stressful situations before the experiments is carried out. The animals were always deeply anesthetized before stroke is induced.

The animal studies were approved (No. S17-10, S38-10, S73-10, S 136-10, S. 199-10) by the regional ethics committee for animal experiments according to the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health (NIH publication # 85-23, revised 1985).

5. Results

5.1 Stroke induces an increased NSCs proliferation in SVZ

The control versus diabetic mice (HFD) did show a significant increase of the NSCs proliferation in the ipsilateral hemisphere subsequent to stroke in the SVZ [ mean = 789 Ki67+ cells in vehicle control vs. mean = 348 Ki67+ cells in vehicle HFD mice: p<0,001 data are presented as mean values ±SEM] (figure 4). However the effect was also present in the contralateral hemisphere to stroke in SVZ [ mean = 881 Ki67+ cells in vehicle control vs.

mean = 419 Ki67+ cells in vehicle HFD mice: p<0,001 data are presented as mean values

±SEM] (figure 5).

5.2 Ex-4 increases stroke-induced stem cells proliferation in SVZ

To investigate the effect of Ex-4 on NSC proliferation, Ki67-expressing cells were counted in the ipsilateral SVZ and contralateral hemisphere by using stereological methods ( see

methods). Ex-4 treatment did induce an increasing effect of NSC proliferation in normal mice [ mean = 1154 Ki67+ cells in Ex-4 normal mice vs. mean = 464 Ki67+ cells in Ex-4 HFD :

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p<0,001 data are presented as mean values ±SEM] in ipsilateral to stroke.(figure 4).The number of Ki67+ cells also increased in contralateral to stroke in normal mice [ mean = 1316 Ki67+ cells in Ex-4 normal mice vs. mean = 529 Ki67+ cells in Ex-4 HFD : p<0,001 data are presented as mean values ±SEM] (figure 5) but not in the diabetic mice although a trend towards an increase of NSC proliferation was recorded in the Ex-4 treated mice [ mean = 464 Ki67+ cells in Ex-4 HFD vs. mean = 348 Ki67+ cells in vehicle HFD mice: p>0,05 data are presented as mean values ±SEM] in ipsilateral to stroke (figure 5).

5.3 Neuroblast production did not altered by Ex-4 in normal mice

In order to determine whether Ex-4 treatment modulated neurogenesis after stroke in normal healthy mice, we used the specific neuroblast marker doublecortin (DCX) in

immunohistochemical (IHC) quantitative experiments. The results in figure 6, show that no significant change in DCX-positive cells was seen in normal mice [mean = 33 DCX cells in vehicle normal vs. mean = 29 DCX cells in Ex-4 normal mice: p<0,05 data are presented as mean values ±SEM] in ipsilateral to stroke.

Figure 4:NSC- proliferation increase in normal versus HFD mice in the ipsilateral hemisphere to stroke SVZ. The number of Ki67+cells. One way ANOVA followed by Tukey's multiple comparisons test was used to evaluate differences between the groups. Differences were considered significant of P value is shown by *P<0.05, **P <0.01 and ***P<0.001 +/-SEM.

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10 Figure 5: NSC- proliferation in normal mice versus HFD, with PBS or Ex-4 treated in the

contralateral to stroke SVZ .The number of Ki67+cells. One way ANOVA followed by Tukey's multiple comparisons test was used to evaluate differences between the groups.

Differences were considered significant of P value is shown by *P<0.05, **P <0.01 and

***P<0.001 +/-SEM.

Figure 6: Neuroblast production in normal mice treated with Ex-4 versus PBS, DCX marked cells was quantified in the striatum. Analysis the unpaired t-test was used to evaluate

differences between the groups. p<0,05 data are presented as mean values ±SEM .Differences were considered significant of P value is shown by *P<0.05, **P <0.01 and

***P<0.001 +/-SEM.

6. Discussion

Stoke has led to a large financial burden on the health system worldwide, because of the long- term disability and mortality connected with the condition[3]. In the present study, we show that the proliferation of stem cell increased after stroke in normal mice with Ex-4 (see figure 4 and 5), in a preclinical setting that mimics the clinical situation. A vast research in the past few years has also investigating whether the regulation of this process could be

therapeutically useful for the treatment of stroke.

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Currently there are still no pharmacological neuroprotective treatments against stroke even though recent study suggest treatments with peroxisome proliferator-activated receptor- gamma (PPAR-gamma) agonists has neuroprotective properties [39]. However r-TPA is the only method that is partially effective against stroke if given within 3-4hours from the onset of stroke[7]. Since the majority of the patients with ischemic stroke do not reach the hospital in time or have counter indications to receive this treatment, r-TPA can be applied to approx 10% of stroke patients[40,41]. Although r-TPA therapy allows blood reperfusion after stroke it does not provide neuroprotection per se. Thus, there is a very high medical need for the identification of new effective drugs against stroke.

Ex-4 is a GLP-1 receptor agonist used in the clinical treatment against T2D (see Intro). Even though the drug has its primary use against T2D, Ex-4 can induce histological and functional recovery in animal models of neurological diseases such as Alzheimer, Parkinson and

Huntington diseases (26, 32,42). Furthermore, earlier studies have shown that pretreatment with Ex-4 gives a positive effect on cell survival and plasticity after stroke. In figure 5 we also improve the effect of Ex-4 with the statistically significant increased number of the NSC:s, even though Ex-4 treatment initiated after stroke. In addition Ex-4 can reduce the size of the ischemic volume in animal models of stroke [30,31,43,44]. Of interest for this project data from other studies indicates that increased NSC:s proliferation suggest increased

neurogenesis. Ex-4 has been shown to stimulate neurogenesis in normal non diabetic rodents as well as to promote cell proliferation in PC12[26,31]. Since T2D patients present decreased recovery after stroke, we hypothesized that this could be in part explained by decreased neurogenesis after stroke. If so, the stimulation of decreased neurogenesis in T2D could be therapeutically useful, unfortunately the result from this project figure 4 and 5, shows that Ex- 4 did not increase the number of proliferating stem cells in diabetic mice.

The major limitation in the development of neuroprotective therapeutic strategies against stroke is contingent upon how quickly neurons die " time is brain". Although several pre- clinically successful drugs has been shown to prove effective against stroke in animal models, they all failed so far in clinical trials once tested on patients [7,42,45].The reasons behind this occurrence may be various. To commence with, the animal model may not mimic the situation of a stroke patient well enough. Several drugs have been tested for their potential efficacy in young healthy animals whereas stroke patients often are old and with

comorbidities such as diabetes, obesity and hypertension [45]. In addition the doses of the pharmacological intervention might also had an enormous part in the difference between preclinical and clinical studies, since candidate drugs were often tested for their potential efficacy at doses hundreds time higher than the one tolerated in man [45].In the end as mentioned before "time is brain" and the time of the pharmacological intervention may not mimic the reality of a stroke patient who arrives to the hospital several hours after stroke time, even so in the pre-clinically trial the pharmacological intervention often starts minutes after stroke or at the time of stroke[45].

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In this study of the potential effect of Ex-4 on NSC proliferation in stroke, we tried to

minimize these factors mentioned above aiming at simulating the clinical situation as much as possible. To do so, we used obese middle-aged mice with T2D since this type of patients has an significance higher risk to develop a stroke when compared to non-diabetic individuals [14]. Under stroke low glucose due to decreased blood flow may further impair brain function and risk neuronal survival. Furthermore, preclinical studies have also shown neuroprotective actions mediated by GLP-1R agonists independently on the regulation of glycemia making Ex-4 a promising pharamacological target for the neurological disorders present in diabetic patients.

Moreover, the Ex-4 treatment was initiated 1,5 h after stroke, thus trying to mimic the urgent situation of a stroke patient receiving a pharmacological treatment in the ambulance. Finally, we gave Ex-4 at the used clinical dose to treat T2D patients, see material and methods. By mimicking comparable situations as much as possible we wished to reduce the risk that our studies in rodents will not be successful once repeated in humans.

Earlier results of the same mice that also were used for this thesis, showed that Ex-4 was neuroprotective at both 1,5 hour and at 3 hours after stroke, in the whole brain[46]. One previous study by Teramoto et al. has tested the hypothesis that Ex-4 was efficacious against stroke once given 1 or 3 hours after stroke [30]. While the authors showed anti-stroke efficacy of Ex-4 1 hour after stroke, they were not able to record a positive effect of the drug at 3 hours after stroke. However, it has to be noted that the read out of this study was based on the

quantification of the stroke volume. Although this assessment is commonly used in preclinical stroke research, it may not be as precise as the quantification of surviving neurons based on stereological counting [37,38] that was performed in Darsalia et al [46]. Therefore the result is promising from this study since quantification of proliferating cells were based on

stereological counting in this study, which is more specific and the immunohistochemistry method is the most commonly used method for this endpoints and research, see method. What is also notable is that the earlier study from Teramoto et al. used a very high dose of Ex-4 ( up to 100 times the dose used in clinical settings for T2D patients) while Darsalia et al [46]

achieved anti-stroke efficacy, with a clinical dose of Ex-4 that produces minimal side effects in T2D patients. Since the same mice were used as in the Darsalia, we could also note the effect of proliferation with clinical doses. Finally the study of Teramoto was performed in young adult mice with no comorbidities such as T2D or hypertension. On the contrary, in addition to employ obese and T2D mice, Darsalia et al [46] study was performed in middle- aged mice. Aging is a very high risk for stroke and it has been estimated that the risk of having a stroke more than doubles each decade after the age of 55 [47]. The experimental design of this study was carefully chosen in many point of view, as mentioned above. What could be improved is the actual staining, where the dilution of the antibody and proliferating marker can affect the staninig, but also the dyeing time can play a role. In this way, one could optimize the immunohistochemistry staining. Until now, the principal method of studying neurogenesis has been to inject either tritiated thymidine or 5'-Bromo-2-deoxyuridine (BrdU) intraperitoneally followed by autoradiographic or immunohistochemical detection methods respectively. However, such exogenous markers may produce toxic effects[48]. Or

endogenous marker Ki67 a nuclear protein expressed in all phases of the cell cycle except the resting phase, since BrdU can be incorporated into DNA only during the S-phase of the mitotic process, whereas Ki-67 is expressed for its whole duration. Ki67 provides therefore

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more evidence to be used as a marker of proliferation in the initial phase of NSC:s proliferation.

More to the aim, a number of studies have demonstrated that the brain possesses a limited regenerative capacity following injury. These investigations have mostly evaluated neuronal production in the adult brain following injury[49,50]; Although encouraging, the amount of cell replacement is limited. However, by understanding the adaptive mechanisms that occur in the SVZ after injury, therapeutic strategies can be designed to achieve a more significant level of regeneration after CNS damage.

The result showed stroke induces an increased NSC proliferation in normal versus HFD mice in the SVZ of the ipsilateral hemisphere. The effect off increased neurogenesis is also present in contralateral to stroke hemisphere. Additionally, lately data indicate that stem cell are more extensive than once believed and exist at multiple sites along the entire ventricular system, consistent with the potential for widespreadneurogenesisand gliogenesis in the adult brain, particularly after injury[50]. Diabetic patients show decreased recovery after stroke in comparison with non diabetic individuals. Increased adult neurogenesis after stroke has been, as mentioned above, proposed to play a role in the partial recovery of patients suffering from stroke. The results presented here indicate that this process is impaired by diabetes, since the proliferation was decreased in HFD mice. The impairment of adult neurogenesis after stroke in diabetic patients could play a role in the decreased recovery after stroke in diabetes. Recent pre-clinical studies have also showed impaired adult neurogenesis in diabetes [51,52].

In addition, Ex-4 response (control versus Ex-4) was statistically significant only in normal and not in HFD, therefore results of this thesis show that the activation of GLP-1R by Ex-4 increased stroke-induced NSC proliferation in normal but not in diabetic mice. Several reports have demonstrated that high susceptibility to stress and elevated corticosterone levels are detrimental to neurogenesis and contribute to neuronal loss. These features are common in some types of depression, diabetes, and aging processes[53]. The effects of Ex-4 have also been studied using a global ischemia model on gerbils [53]. The data showed robust neuroprotection induced by Ex-4. However, as for the study from Teramoto et al, the experiments were conducted on young healthy animals not simulating the status of the

majority of stroke patients (aged and with co morbidities). Furthermore, the administration of Ex-4 was given 2 hours prior to stroke and directly after reperfusion of the stroke, hence not bearing clinical relevance. The data suggest that if this pharmacological treatment could be beneficial in non-diabetic patients suffering from stroke, then we still need find other drugs that could be useful in diabetic patients suffering from stroke.

However, The data of DCX staining, see figure 6, showed no significant change in DCX+

cells in striatum which may be because the mice were sacrificed too early after the stroke, so the time was not enough to migrate to striatum[54,55].

I believe that it is of great significance that the proliferation was increased even if the Ex- 4 treatment was initiated 1,5 hours post-stroke. That will give us relevant data for clinical perspective. Following these results, it would be interesting to determine if the effect of increased proliferation of stem cells also can be promoted after more than 3 hours in normal

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mice. By clearly defining the therapeutic window of the Ex-4 treatment, these studies will hopefully put the basis to design a clinical strategy with practical chances of success.

In conclusion our findings hold great clinical relevance. In the future it would be of great interest to conduct studies aimed at determining whether Ex-4 would hold a significant anti- stroke effect in other groups at high risk of stroke (e.g. hypertensive patients) other than obese and T2D patients. The cellular and molecular mechanisms at the basis of neuroprotection induced by Ex-4 have been poorly investigated. The traditional stroke treatments may pose a greater risk of hemorrhage in diabetic patients due to their weakened vascular system when compared to healthy individuals [56] Therefore, when developing stroke treatments for diabetics, the importance should be focused on substances that ideally will strengthen the cardiovascular system and protect the nervous system at the same time.

But most likely after all the data above, I think Ex-4 strongly effects the nerve cell through cell fusion between the nervcell and blodcell to obtain essential protien so the neuron can survive after stroke. Previous study have shown that specific inflammation in the brain increases fusion between blood cells and nerve cells[57]. Thus another important goal for the future research in this field will be to identify the mechanisms at the basis of the effects mediated by Ex-4.

.

7. Acknowledgements

I would like to thank my supervisor Dr. Cesare Patrone for providing continuous support and discussions as well as feedback throughout the course of this project You always believed in my academic ability, even on days when I was a bit “lost”. Thank you for providing an excellent research environment, with valuable and constructive discussions. Thank you!. I would like to thank Dr. Vladimer Darsalia for teaching me the numerous methods that were needed in this project as well as providing experimental assistance and support. I would also like to thank Dr. Shiva Mansouri, Mohamed Eweida and Grazyna lietzau for providing useful discussions about the different aspects of the project and assisting me in the lab whenever help was needed. Thank you Jeanette Lundblad-Magnusson for all the support you gave me.

I would like to thank my internal supervisor Eewa Nånberg, and my examinatior that always helped me when I needed, I'm so thankful Lars Järnström,

Finally, to my wonderful and loving family, none of this would have been possible without you. I cannot put my feelings and my gratitude into words. Thank you for believing in me and encouraging me. Thank you my beloved husband for being there through thick and thin, you always give me strength at difficult times. I dedicate this thesis to you, my family.

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54. Moraga A, et al. Aging increases microglia proliferation, delays cell migration, and decreases cortical neurogenesis after focal cerebral ischemia. J Neuroinflammation. 2015 May 10;12(1):8 55. Lin R, et al Neurogenesis is enhanced by stroke in multiple new stem cell niches along the

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Books used for stereology methods:

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9. Appendix

9.1 Number of Ki67+ cells in each sample

Table.1

id Normal ipsi Normal contra

1 633 803

2 896 901

3 810 747

4 953 819

5 857 1007

6 682 923

7 799 957

8 683 892

Table.2

Id HFD ipsi HFD contra

32 246 347

33 345 315

34 218 567

C1 373 402

C2 412 460

C3 322 382

C4 377 383

C5 331 398

C6 506 513

Table. 3

Mice Id Ex-4 normal ipsi Ex-4 normal contra

18 1173 1476

19 1253 1582

21 1223 1586

23 1029 903

30 1036 1132

31 1362 1355

32 1059 1258

7 1097 1239

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19 Table. 4

Mice Id Ex-4+ HFD ipsi Ex-4 + HFD contra

16 518 557

17 433 661

18 239 369

19 456 482

21 576 588

22 419 469

23 472 582

24 554 515

25 511 535

9.2 Number of DCX+ cells in each sample

Table. 5

Mice Id

DCX- NORMAL

1 15

2 39

3 36

4 45

5 36

6 22

7 30

8 39

Table. 6

Mice Id

EX-4 DCX

18 25

19 27

21 26

23 33

30 15

31 34

32 43

Stroke-induced stem cells proliferation in normal versus diabetic mice and pharmacological regulation