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A Possible Rejuvenation of Neurons Using Human Umbilical Cord Blood Through the Akt Pathway by Vijay Mehta A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree of Bachelor of Arts in Neurobiology Under the sponsorship of Dr. Alfred Beulig Sarasota, Florida May, 2011
Acknowledgements There are a number of people I need to thank for he lping me to find a thesis topic and implement it. Thank you to Dr. Alison Willing at US F, Tampa, Center of Excellence for Aging and Brain Repair. I will be forever grateful for the opportunity you gave me to work in a fantastic lab, and in an area of research that is important in medicine. The time I spent at the lab was both educational and very en joyable. I also have to thank you for giving me the chance to carry out the experiments f or my thesis, and then helping me to construct my thesis. Thank you to Dr. MD Shahaduzzman, for spending time with me in going though the experiments and organizing every step. The time I spent with you in other projects also helped me gain a better understanding of resea rch with human umbilical cord blood. Thank you to Jason Golden for educating me on lab t echnique, helping me through technical aspects of the experiments, and editing m y thesis. I am much indebted to Dr. Alfred Beulig for all his hard work in the process of mentoring me through the drafting of my first scien tific work. Thank you to Dr. Katherine Walstrom and Dr. Leo Demski for being on my baccalaureate committee, and to all three of you for the informative classes. Thank you to the travel and research committee and the council of academic affairs for the grants and travel award, which made this thesis possible.
Last, but not least, thank you to family and frien ds; my parents Bina and Narendra for helping me get here, my brother Jayesh and my r oommate Nick for the constant encouragement.
Table of Contents Title i Acknowledgements ii Table of Contents iv List of Figures and Tables vi List of Acronyms vii Abstract x Chapter 1: Introduction 1. Stem cells 1 2. Collection of CB 1 3. CB Composition 2 4. CB applications 4 5. Stroke 8 6. Ischemic Stroke Pathophysiology 9 7. Stroke treatment with CB 12 8. Akt (PKB) pathway 18 9. MAPK Pathway 24 10. Akt+MAPK in Stroke
i. Akt 27 ii. ERK Promoted Cell Survival 28 iii. ERK Induced Cell Death 29 11. Akt +MAPK + CB in stroke 30 Chapter 2: Method 1. Akt inhibitor 33 2. MAPK inhibitor 35 Chapter3: Results 1. Akt 36 2. MAPK 41 Chapter 4: Discussion 46 References 51
List of Figures and Tables: Figure1: An outline of the cell in stroke condition s 10 Figure 2: Interaction of Ras in Akt and MAPK pathwa y 19 Figure 3: Activation of Akt 20 Figure4: Upstream and downstream proteins of Akt 23 Figure5: An outline of the MAPK pathway 25 Figure 6: Image of a 6 well plate and insert 35 Figure 7: Images of neurons in Normoxic condition t reated with Akti 37 Figure 8: Bar graph of cell viability in normoxic c onditions 38 Figure 9: Images of neurons on OGD conditions treat ed with Akti 39 Figure10: Bar graph of cell viability under OGD con ditions 40 Figure 11: Images of cells in normoxic condition tr eated with MAPKi 42 Figure 12: Images of cells in OGD conditions treate d with MAPKi 44 Table 1: Cell viability of neurons in normoxic cond ition treated with MAPKi 43 Table 2: Cell viability of neurons in OGD condition treated with MAPKi 45
List of Acronyms AMPA: 2-amino-3-(5-methyl-3-oxo-1,2oxazol-4-yl) propanoic acid BBB: Blood brain barrier BM: Bone marrow CB: Human umbilical cord blood cells. CINC1: cytokine induced neutrophil chemoattractant 1 CPT: Camptothecin CREB: cAMP response element binding protein DMEM: Dulbeccos modified eagle medium DRG: Dorsal root ganglion DTG: 1, 3, di-0-tolylguanidine ERK: extracellular signal-regulated kinase EGF: Endothelial growth factor GMCSF: Granulocyte macrophage colony stimulating factor Grb2: Growth factor receptor-bound protein-2 GSK3: Glycogen synthase kinase 3 GVHD: Graft versus host disease HLA: Human leukocyte antigens HUCB: Human umbilical cord blood IAM: Intra-arterial injection of CB with mannitol IC: Intracerebral ICAM1: Intercellular adhesion molecule -1 ICE: Interleukin1-converting enzyme IGF1: Insulin growth factor1 IL: Interleukin
MAPK: Mitogen activated protein kinase MAP2: Microtubule associated protein-2 MCAO: Middle cerebral artery occlusion MCP1: Monocyte chemoattractant protein MEK: Mitogen activated kinase kinase MK: MAPK-activated protein kinase MNC: Mononuclear cells MNK: MAPK-interacting kinase MSC: Mesenchymal stem cells NeuN: neuronal nuclei NFB: nuclear factor kappa-light-chainenhancer of activated B cells NGF: Nerve growth factor NK: Natural killer cells NMDA: N-methyl-D-aspartic acid NOS: Nitric oxide synthase NWSM: Norcodia water soluble mitogen OGD: Oxygen and glucose deprived PARP: poly (ADP-ribose) polymerase PC12 :Pheochromocytoma of rat adrenal medulla PDGF: platelet derived growth factor PDK-1: Pyruvate dehydrogenase kinase -1 PI3K: Phosphatidylinositol-3 kinase PIP: Phosphatidylinositol 3-phosphate PIP2: Phosphatidylinositol 3, 4-bisphosphate PIP3: phosphatidylinositol 3, 4, 5-triphosphate PKA: cAMP dependent protein kinase PKC: Protein kinase C PH: Pleckstrin homology
RSK: ribosomal S6 kinases SCG: Superior cervical ganglion TGF1: Transforming growth factor1 TIA: Transient ischemic attack TNF: Tumor necrosis factorTOR: Target of rapamycin
Abstract: In the last 20 years the use of Human Umbilical Cor d Blood Cells (CB), specifically the mononuclear cellular fraction of H UCB (MNC), has become an important therapy in multiple diseases, such as leukemia, and tissue degeneration. In the last 10 years the use of CB has been expanded to include ne urodegenerative diseases and traumatic brain injuries. Its use in in vivo studies of stroke and traumatic brain injury has resulted in more than 80% recovery of brain cells i n damaged areas. Studies have elucidated that the peripheral response, the cells outside the brain, is responsible for much of the neuronal death in strokes. The addition of C B at specific time points therefore allows it to intercede with the peripheral response and promote the production of antiinflammatory cytokines such as IL-10. This experime nt examined the potential cell survival pathways that mediate CBs therapeutic eff ects on neurons. The two main pathways are the phosphoinositide 3-kinase/Akt (PI3 K/Akt), and mitogen activated protein kinase/ extracellular signal-regulated kina se (MAPK/ERK). Neurons were collected and used in oxygen and glucose deprived c onditions (OGD) which simulate the ambient conditions in ischemic stroke. Control grou ps were exposed to normal levels of oxygen and glucose. Each condition was treated with either Akt or ERK inhibitor as well as co-culture with CB. Results demonstrated that bo th pathways are significant in protecting neurons during exposure to OGD condition s, but the Akt effect was more significant in normoxic conditions. In OGD conditio ns cell viability for neurons with CB was at 797.26. This decreased significantly (p<0.0 1) to 46.72.02 when treated with CB and Akt inhibitor. The same conditions, but with MA PK inhibitor showed a significant (p<0.05) decrease in cell viability from 80.61.91 to 53.81.67. Therefore this study
demonstrated that the Akt pathway is the more influ ential pathway in CB ability to induce a neuroprotective effect. Dr. Alfred Beulig Division of Natural Sciences
Introduction: 1. Stem cells In recent years stem cell therapy has become a majo r field in regenerative medicine. Stem cells are progenitor cells which are able to form any part of the body. The most well-known cells that are able to do this are embryonic stem cells; which proliferate to large numbers then differentiate to form organs, afterwards losing their totipotent abilities. For a long time embryonic stem cells wer e viewed as holding great promise for advancement of health care. However, there are ethi cal problems in trying to obtain embryonic stem cells as it involves termination of an embryo. Also, due to the early stage in research, the smaller chance of cell rejection c an be offset by the resulting teratomas, as a result of uncontrolled cell replication. There fore the high costs of treatments, around $20,000 in transplant centers such as Germany, can often go to waste as treatment is ineffective for the patients. As a result, other fo rms of stem cell production are being investigated, including induced pluripotent stem ce lls and human umbilical cord blood cells (Cairo and Wagner, 1997). Human umbilical cord blood (HUCB) was successfully used by Gluckman et al. (1989) for bone marrow transplant. Since then, it h as been used as an experimental treatment for malignancies, hematological disorders inborn errors of metabolism, degenerative disorders, injuries, and a wide variet y of other medical conditions. 2. Collection of CB:
Collection of HUCB can be done in utero or ex utero although in utero would have ethical issues as the mother and child would b e in an uncomfortable position. The technique is very safe, and involves cannulation of the umbilical vein. The cleaner method for extracting HUCB is after a cesarean sect ion as there is decreased contact with the skin and vaginal flora. Collection must be done in sterile conditions to make sure there are no traces of infections. The family histo ry of disease as well as the blood type of the fetus is recorded when banking the blood so tha t future matches can be made as accurately as possible (Pahwa et al., 1994). This will help to prevent graft versus host disease (GVHD) by matching the human leukocyte anti gens (HLA). The blood is filtered through three bags containing anti-coagulants follo wed by two centrifugations, following which the red blood cells are removed. Studies have shown that the amount of maternal blood present is less than 1 in 10000. Of the CB cl inical trials thus far, there have been no clinical immune reactions to the maternal blood sug gesting that the treatment is welltolerated (M-Reboredoa et al., 2000). 3. CB Composition: The mononuclear cell (MCN) fraction of CB includes many immature cells that are found in the adult immune system, such as hemat opoietic stem cells (HSC), mesenchymal stem cells (MSC), granulocytes, erythro cytes, macrophage, megakaryocytes, T cells, and B-cells. CD 34+ hematopoietic stem cells are progenitor cells which can be induced into divide and produce various other blood cells such as erythrocytes, white blood cells, B-cells, or T-cell s. Proliferation of CD 34+ cells is possible through addition of IL-11, stem cell facto r and granulocyte macrophage colony
stimulating factor (GMCSF), and may provide enoug h cells to be used in adults (van de Ven et al., 2005). The Human Umbilical Cord Blood Mononuclear fraction includes CD4+ helper T cells and CD16+ natural killer cells (NK), which produce tumor nec rosis factor-, IL-4, IL-6, and interferon CB is 10-1000 times less reactive to antigens tha n adult peripheral blood in regard to proliferation of cytotoxic T lym phocytes (Harris et al., 1994). Indeed, allogenic bone marrow transplants featuring CB tran sfusion have a much lower incidence of rejection and immunogenic response from the reci pient. Although the percentages of NK cells in CB and adul t peripheral blood are comparable (Harris et al., 1994), it was later foun d that the NK cells are suppressed by factors during pregnancy and may be present in the serum after the blood is taken. The authors found that freeze-thawing removed many of t he factors that inhibited the NK cells enabling functionality comparable to adult NK cells. In particular, in vitro addition of IL-2, IL-12, and IL-15 activated the NK cells to a state comparable with the adult forms. The immature nature of CB, and in particular the suppression of NK cells and Tcells, gives the CB the property of lowered immunog enicity, making it desirable as a potential treatment (Dominguez et al., 1998). Although recent research (Chunduri et al., 2008) h as shown that CB poses a risk of GVHD, the effect with the MNC is likely to be su ppressed for at least some time. The authors examined CD34+ CB cells alone and determined that there is a much greater activity and concentration of HLA-DR, an antigen th at promotes T-cell activity. However, the mononuclear fraction required repeated stimulation over four weeks before
acquiring high enough levels presenting with enough HLA-DR to activate T-cells capable of lysing cells. This suggests the possibility of c hronic GVHD through the aggregation of HLA-DR and T cells. 4. CB Applications: CB was first used to treat Fanconis anemia in a ma le child about twenty years ago using the umbilical cord blood of the boys sis ter. The treatment was successful as 20 years later the person is still alive and has compl ete hematological reconstitution. Since then, twenty thousand CB transplants have been perf ormed (Gluckman 2009). Due to the presence of hematopoietic stem cells, the use of co rd blood was preferred over the use of bone marrow. Also, treatment of acute leukemia in m ore severe patients showed greater success using CB rather than bone marrow treatment, even though the CB was not HLA matched. Examination showed a decreased incidence o f acute GVHD, while incidence of chronic GVHD remained the same (Rocha et al., 2001) Again, however, studies have shown a delayed GVHD response to CB; and there is e ven less incidence of GVHD if HLA matching of two antigens is performed (Eapen et al., 2007). CB use is limited by the small volume (20-75 mL) of blood samples harvested from the umbilical cord after birth relative to bon e marrow samples harvested from the femur. Treating adult patients or even larger-sized children is therefore more problematic with CB, and future treatments may require culture and passaging to produce cells in sufficient quantity.
Among the stem cells included in CB are mesenchymal stem cells (MSC); the properties of which have been explored by inducing the MSCs to differentiate into pancreatic cells, bone, muscle, and neurons. In par ticular, MSC-derived pancreatic islet cells have been able to produce insulin. Therefore this could be a future therapy for patients that have kept their own CB as there would be no issue of GVHD (Phuc et al., 2011). The differentiation of the MSC cells into ot her cell types has suggested its possible therapeutic use in diabetes, arthritis, ne urological disorders and myocardial infarction (Gluckman 2009). Clinical trials have been approved for CB in the tr eatment of diabetes mellitus type 1 which is characterized by the autoimmune des truction of insulin producing pancreatic beta cells. The trials were conducted wi th a patient cohort whose inclusion criteria specified the prior banking of autologous cord blood. Preliminary results have shown no adverse effects of the treatment as well a s a significant decrease in the requirement for supplementary insulin. Although mor e testing is necessary, the therapeutic effects have been clearly demonstrated (Haller et al., 2008). However, the investigators emphasize the caveat that the treatme nt requires banked autologous CB, and express concern over whether enough pancreatic cell s can be produced from the small volume of CB harvested from the umbilical cord (Phu c et al., 2011). CD34+ cells have shown the potential of producing endoth elial cells and vasculature. Experiments have shown that when the c ells are injected into rats with damaged lung tissue, CD34+ cells differentiate into both alveolar type I and II cells. The type II cells act as alveolar progenitor cells whic h can form more type II and type I cells
n (De Paepe et al., 2011). It is thus seen that vario us cell populations within the CB can be used to treat a variety of diseases. In particular, the potential for the CB cells to regenerate neurons has been widely investigated. Since the late 1990s there has been a vast amount of research perfromed examining CB as a potential treatment for trauma, s troke, and neurodegenerative diseases. Early research looked at the potential of the CB for forming nascent neurons through differentiation. Stimulating CB with neural growth factor, researchers found that some cells exhibited many neuronal markers such as Musashi-1, -III-tubulin, pleiotrophin, and neuronal nuclei (NeuN). For insta nce, -III-tubulin is a unique form of tubulin found in neurons, showing that certain cell s in CB had differentiated enough to produce this marker (Sanchez-Ramos et al., 2001). A dditionally, the percentage of cells expressing these markers increased from the origina l CB samples following stimulation. CB was also injected into rats and mice with in vivo models of stroke or trauma. Initial results showed that the CB cells migrated t o the site of injury (Lu et al., 2002). Later, immunocytochemistry showed that the cells pr oduced neural markers NeuN and MAP-2 as well as the astrocytic marker GFAP, while some even integrated into the surrounding tissue at the site of injury. The subje cts had improved significantly compared to controls with the majority of the cells migratin g to the brain around the site of injury. Conditioning the CB is an important aspect of maxim ising its therapeutic effect and this was shown by culturing CB in Dulbeccos Modified Ea gle Medium (DMEM) +fetal bovine serum or Neurobasal +retonoic acid +neuronal growth factor. After incubation in this media, the cells were injected into rats. Twen ty percent of the CB cells survived and
were spread amongst the site of injection, cortex, and corpus callosum. Some of these showed differentiation through markers such as -III-tubulin (Zigova et al., 2002). Following these investigations, Willing et al. (200 3) compared intravenous and intrastriatal injection of CB in middle cerebral ar tery occlusion (MCAO) stroke-model rats. The results were very telling as the intraven ous injections showed better recovery when compared with controls as measured by in histo logical examination and behavioural testing. The lack of CB found in the br ain, and the better results from intravenous compared to intrastriatal injections of CB led to the hypothesis that the effects of CB were peripheral as opposed to local, in which case the stem cells would grow into new neurons. Examining the MNC fraction o f the CB, i.e. white blood cells, and recognizing that monocytes and lymphocytes aggr egate around the site of injury during stroke or trauma; it was proposed that the C B exerts its therapeutic effects via the immune system. The hypothesis of a peripheral effect was further v alidated when a study compared intracerebral injection (IC) and intra-art erial injection (IAM) potentiated with the addition of mannitol, which acts as a BBB perm eabilizer. Both the IC and IAM groups showed a reduction in infarct size. However, the histochemistry showed no sign of CB in the brain with IAM, while such presence wa s seen in the IC group. Therefore, the reduction in infarct size is hypothesized to oc cur because of the secretion of neurotrophic factors from the CB (Lobel et al., 200 3). Other investigations of neural regeneration focus o n spinal injuries. To investigate this, spinal cord injuries were surgically induced in rats and CB was injected in the tail
vein. Within three weeks there was a significant di ffence in the mobility of the rats. The post-morterm highlghted stem cells aggregated to th e site of injury, but instead of showing proliferation of neurons and glial cells, r ecovery is thought to be through factors released by the cells (Saporta et al., 2003). Previ ous research by Coumans et al. (2001) showed that the regeneration of the spinal cord imp roved after addition of stem cells and neurotrophic factors 2-4 weeks after the trauma. Th e researchers also demonstrated better growth of axons by adding brain-derived-neurotrophi c factor and neurotrophin-3. The presence of the neurotrophic factors was crucial in the regenereation of the spinal cord as was the time scale for repair to occur. This is bec ause the inflammatory response to the injury decreases after 2 weeks. 5. Stroke: Stroke is the third leading cause of disease in the US; a person has a stroke every 40 seconds. A stroke may occur through clots obstru cting the flow of blood through the blood vessels (ischemia) or through the blood vesse l bursting causing the release of blood into an area of the brain (hemorrhage). Ischemic st rokes are the most common and account for around 87% of strokes, while 10% are ca used by intracerebral hemorrhage and 3% are from subarachnoid hemorrhage. Strokes va ry in their duration with transient ischemic attacks (TIA) symptoms lasting for less th an 24 hours. This form of stroke accounts for 15% of strokes and is thought to lead to recurrence in many individuals with 1 in 4 dying within the first year. As well as bein g the third leading causes of death it is also the most costly as a result of disability issu es. The estimated cost for stroke is 68.9
r billion per year which includes both costs to the i ndividual (direct) and to society (indirect) (Lloyd-Jones et al., 2009). The need to prevent the disease is therefore as i mportant as finding a treatment that would allow patients to recover from a stroke. Although research has extended for many decades, there has been little success in deve lopment of effective measures in preventing stroke (Hall and Pennypaker, 2010). 6. Ischemic Stroke Pathophysiology In an ischemic stroke, a blood clot, atheroscleroti c plaque, or some form of obstruction occurs in a blood vessel in the brain. As a result, that area of the brain is oxygen and glucose deprived (OGD), which the brain solely relies upon for the production of ATP, ionic balance maintenance, and t he movement of neurotransmitters. The result of the OGD conditions is the depolarizat ion of neurons, and release of excitatory amino acids such as glutamate at the syn apses. This causes the ion channels in the postsynaptic neurons to open resulting in large influx Ca2+, Cl-, Na+ and K+. The lack of ATP means that ATPdependent Na+/K+ pumps cannot exchange the necessary ions and therefore cannot maintain ionic homeostasis in the neurons. The up-regulation of ions in the cells, such as Na+, results in the diffusion of water inside the cell cytolysis, and swelling of the tissue (shown in Figure 1). This is a very significant pathological marker of stroke, and the size of the swelling can often s how how severe the stroke will be (Dirnagl et al., 1999).
Figure 1. A basic outline of the impact of stroke on neurons. The presynaptic terminal releases glutamate because of the depletion of ATP. The postsynaptic terminal is depolarized by glutamate, which causes ion channels to open. The influx of Na+ cannot be corrected as a result of the depletion of ATP. Water moves in through osmosis causing cell swelling. Ca2+ causes mitochondrial damage and reactive oxygen species production which lead to apoptotic mechanisms and activation of the immune system (Dirnagl et al., 1999). Interstitial Ca2+ moves into the surrounding cells where it activate s Ca2+ dependent enzymes such as nitric oxide synthase (NO S). NOS reacts with a superoxide anion which results in the formation of peroxynitra te. This is a very reactive species which causes damage to the inside of the cell. A ca scade begins in which oxidation within the cells results in radical species which d estroy many organelles including mitochondria. This causes the release of Cytochrome C which then initiates apoptosis (Tibor and Bo, 1998). Within several hours of the occlusion cell death o ccurs for cells exposed to OGD conditions. However, a border area, known as the pe numbra, includes cells not initially exposed to OGD, which nevertheless decay over a lon ger period of time. The time-based decay of the penumbra can vary depending on how sig nificant the occlusion is. The release of Ca2+ causes secondary mechanisms (as in figure 1 above) to initiate and proinflammatory genes to activate. A buildup of the in flammatory molecules interleukin-1
(IL-1), IL-6, and tumor necrosis factor(TNF-) is seen 6-8 hours after stroke (Rothwell and Hopkins, 1995). This induces intercellular adhe sion molecule 1 (ICAM-1), Pselectins, and E-selectins, which allow neutrophils that have crossed the BBB to attach to cells in the CNS. Following the neutrophils are the monocytes which help in clearing much of the debris including the older neutrophils. Concurrently the microglia, which act as the immune cells of the CNS under pathological c onditions, activate and migrate into the penumbral region, where they secrete IL-1, TNF-, NO, and hydrogen peroxide (Giulian 1997). The prevalence of these excitotoxic factors peaks b etween 6-8 hours after which the penumbra also becomes affected. The penumbra is still able to receive some supply of oxygen and glucose and so is able to hyperpolarize and depolarize, resulting in the release of more glutamate and ions. Studies have sh own that cell apoptosis in this region occurs from 24-72 hours following stroke (Endres et al., 1998). It has been thought for many years that the immune response has a crucial role in the extent of damage incurred in stroke. This theor y was supported by the work of Ajmo et al. (2008), who demonstrated that removal of the spleen before stroke decreased infarct size by more than 80%. It is clear that the periphe ral response is associated with macrophage and neutrophil infiltration of the brain as a result of the breakdown of the BBB. The addition of anti-CD11b, a marker for sever al immune cells, at 2 hours after stroke significantly reduced the volume of the infa rct. This is because the antibody stopped the adhesion of neutrophils, monocytes/macr ophage, and larger lymphocyte granulocytes to ICAM-1 (Zhang et al., 1995). The pe ripheral response thus plays a key
role in the determination of infarct volume and lik ely contributes to the activation of microglia, further increasing inflammation. Removal of the spleen also resulted in a decrease in the aggregation of monocyte/macrophage and neutrophils at the BBB. Timing is a crucial factor in stroke treatment. As a result many treatments which have targeted the NMDA and AMPA receptors have fail ed as these treatments need to be started within an hour of the stroke. Blockage of g lutamate-activated Ca2+ channels has resulted in dangerous side effects such as respirat ory depression. The lack of success in these trials can also be attributed to the innate d ifferences between humans and rats. The middle cerebral artery occlusion (MCAO) model of st roke is widely used in animal experiments. However, there is a substantial differ ence in the size of the cortex in humans in comparison to rats. A stroke in humans therefore results in a much greater range of motor function deficits (Hall & Pennypacker, 2010). 7. Stroke Treatment with CB: Research in the treatment of stroke with CB began m ore than ten years ago when Chen et al. (2001) studied the mechanisms in which CB impacted rats with experimentally-produced strokes. In this experiment CB was injected at 24 hours and 7 days post-stroke. The results showed significant di fference between the two with better recovery of movement following injection at 24 hour s compared to 7 days. This was supported by histological analysis which showed mor e CB cells were found at the site of injury when Rats were injected 24 hours after strok e. It is believed that the therapeutic effect of CB is derived from the release of neurotr ophic factors which are able to move through the more permeable BBB following stroke. A study by Belayev et al. (1996)
illustrated that the BBB opens 4 hours after MCAO a nd peaks in terms of permeability at 48 hours. Therefore if it is the neurotrophic fact ors that influence the recovery it would coincide with the influence of CB being greater whe n injected at 24 hours as compared to 7 days. This would relate to the release of proinfl ammatory molecules from the site of injury causing the CB as well as other cells in the immune system to migrate to this area. Following this study and the work by Willing et al. (2003) in comparing the routes of administration many of the same authors l ooked at the effect of CB dosage (Vendrame et al., 2004). CB doses of 104 and 105 had very little difference in behavior and motor function while a dose of 106 and greater had a significant difference. However, there was little change in behavior when the number of cells was greater than 106. On the other hand, analysis of the tissue showed that a do se of 107 cells caused the greatest reduction in infarct size. Just as significant was the fact that CB cells were found in the spleen, but were not found in the brain to any sign ificant extent. In a follow up to this study, the same investigator s studied the effect of CB injection at various points in time after ischemic stroke. This study provided crucial results by illustrating that CB at a concentration of 106 injected 48 hours post-ischemic injury produced the best recovery in the core area of the stroke. These results are significant compared to the article above (Vendrame et al., 2004) in which a dose of 107 at 24 hours post-stroke produced the greatest reduc tion of infarct volume. This combined finding suggests that the initial stroke region is not completely dead and instead of all the cells going through necrosis as a result of inflamm ation, most are going through apoptosis: programmed cell death (Newcomb et al., 2 006). The effectiveness of a dose of
106 CB at 48 hours as opposed to 24 hours is likely to be due to the action of the immune system. The peripheral immune system plays a key role in mo dulating the infarct size from a stroke. The increased levels of IL-6 within the first few days of a stroke are positively correlated with the size of the infarct as well as the problems the person would face at 3 months and 12 months. The presence of IL6 is thought to be influenced by both IL-1 and TNF(Smith et al., 2004). Therefore, understanding the interaction of CB with the immune system early in a stroke could be vital to development of a more effective treatment and improved prognosis after 3 months. In terestingly IL-6 produced at high levels has increased the production of transforming growth factor-beta1 (TGF-1) by Tcells (Zhou et al., 1993). TGF-1 is found in mitochondria, and is an important in inhibiting apoptosis through down regulating apopto tic mechanisms within T cells (Chen et al., 2001). CB is also known to release IL-10, a very potent anti-inflammatory cytokine, as well as endothelial growth factors, an giopoietin 1 and 2, and vascular endothelial growth factor (VEGF) (Pomyje et al., 20 03). T-cells in CB produce IL-10 abundantly, whereas adult T-cells need multiple sti mulations before the production of IL10 reaches similar levels. However, CB T-cells beco me habituated to IL-10 after further stimulation, while adult T-cells continue to increa se levels of the cytokine (Rainsford and Reen, 2002). Therefore, CB could help in promoting more of an immediate antiinflammatory response. Further recognition of the impact of CB on the immu ne response was studied by Vendrame at al. (2005). CB treatment was associated with a significant decrease in the
number of pro-inflammatory cytokines TNFand IL-1. This expression correlated with the decrease in nuclear factor B (NF-B) which is activated by TNFand IL-1. NFB binds to DNA and coordinates transcription of cyt okines during stress. As mentioned previously, anti-inflammatory cytokine IL-10 is ver y important and has been found to reduce the infarct volume in stroke (Strle et al., 2001) and inhibit NF-B. The inhibition by IL-10 results from significant binding to p50, a type of NF-B subunit. The observation may explain the fact that NFB promotes survival in neurons, but ushers a pro-inflammatory response in microglia and astrocyt es which have more p65 subunits (Pennypacker et al., 2001; Driessler et al., 2004). CB treated MCAO rats had upregulated IL-10 protein in the brain associated wit h unchanged levels of IL-10 mRNA, suggesting that the IL-10 is one of the cytokines b eing secreted by cord blood. Flow cytometry also showed a decreased number of CD45+/CD11b+ microglia/macrophage and CD45+/B220+ B-cells in the brain (Vendrame et al., 2005). As d iscussed previously, the microglia become activated in conjunction with the peripheral response and can result in more cell apoptosis through production of glutamate nitric oxide, IL-1, IL-6, and TNFwhich peak 2-3 days post stroke (Mabuchi et al., 20 00). B cells are likely to provide more antigens for the activation of microglia. The impact of microglia is prevalent in many neurol ogical diseases including Alzheimers disease. The marker CD40 is also implic ated in activated microglia (Tan et al., 1999). This is significant as a comparison of monocytes between CB, BM, and peripheral blood (PB) demonstrated a lack of CD40 i n CB while the latter two had some expression. The presence of CD40 causes maturation of B cells and therefore the increased production of IgG or IgA via the influenc e of T helper cells. However, in CB
n the T-helper cells are still immature and even with stimulation with Norcardia water soluble mitogen (NWSM) in vitro there was little production of IgG and IgA (Nagoki et al., 1981). Macrophages also increase activation an d present more CD40 and TNF receptor sites (Sorg et al., 2001). The increased activation of microglia and other imm une cells is likely to be the result of chemokines such as the inflammatory prote ins monocyte chemoattractant protein (MCP-1), cytokine-induced neutrophil chemoa ttractant -1(CINC-1), and intercellular adhesion molecule (ICAM1). MCP-1, unlike t he other two, promotes IL-10 as well as NF-B, and therefore promotes some neuronal survival. M CP-1 is also thought to cause the release of glucocorticoids which suppress pro-inflammatory agents such as IL-1 and TNF(Thompson et al., 2008). However, it also causes m ore macrophages to accumulate, (which remove the neutrophils), but cau ses an increased buildup of these immune cells which invade the BBB (Hall and Pennypa cker, 2010). CINC-1 attracts neutrophils and responds to the increase in IL-1 and IL-6. All chemokines are present after the first 2-3 hours following insult. However the effect of CINC-1 is delayed. There is a rapid elevation of neutrophils in the liver, b ut these cells take a longer time period to migrate to the brain (Campbell et al., 2003). As me ntioned previously, adhesion molecules ICAM-1, E-selectin and P-selectin are up regulated by NF-B after which they allow adhesion of leukocytes which increase brain i njury. Inhibition of ICAM-1 is known to prevent ischemic brain damage in the transient s troke model as opposed to the permanent MCAO which has been used in the majority of the CB studies (Vemuganti et al., 2004).
Interestingly the peak of pro-inflammatory molecule s MCP-1, CINC-1, and ICAM-1is around 48 hours which coincides with CB in duced recovery of the stroked area by Newcomb et al. (2006). It is therefore like ly that the same factors induce the immune cells in CB to aggregate in this area allowi ng them to have the effect mentioned. However, even this 48 hour mark is liable to change both with rats and humans. As people age, the ability to regenerate tissue decrea ses significantly, and apoptotic mechanisms become more prevalent. Studies comparing 3 month and 20 month old rats with experimental stroke demonstrated that the core area had greater cell death in adults. On the other hand there was a delay in growth promo ting factors such as c-jun. There was also an increase in the amount of toxic factors suc h as the C-terminal fragment of amyloid precursor protein two weeks after stroke in 20 month rats (Petcu et al., 2008). It is therefore likely that the injection of CB would need to occur earlier in older rats. This may correlate with large differences in time with r espect to potential therapeutic effects in humans and reasons why many other drugs have fai led. So far CB is the only therapy that has provided pro tection for neurons when used in cell culture. However, in vivo, 1, 3, di-0-tolyl guanidine (DTG) injected 24 hours after stroke has shown a reduction in infarct volume simi lar to CB at 48 hours. DTG is a sigma receptor agonist and acts as an anti-inflammatory a gent which interferes with intracellular calcium signals. In vivo it decreases the activatio n of microglia/macrophage and therefore decreases the production of NO (Ajmo et al., 2006). However, in vitro experiments have shown that it has no effects in terms of neuroprote ction whereas CB shows the same neuroprotective ability. Therefore the two substanc es are likely to use different mechanisms to protect the neurons, although both se em to impact the immune system
(Hall et al., 2009). So far work has been done as f ar as looking at the pathway on which CB has its effects, but no definitive pathway has b een shown. 8. AKT (PKB) Pathway Akt is a serine/threonine kinase that is expressed in all organisms, and has a very conservative coding sequence. It was first designat ed c-Akt as it was similar to a viral oncogene. It is also similar to the protein kinase C (PKC) family (Bellacosa et al., 1993). It is known to be involved in cell signaling and pl ays a very important role in activating anti-apoptotic pathways. Examination of various neurotrophins including nerv e growth factor revealed that certain factors and pathways they initiate are very important in neuronal survival. Nerve growth factor (NGF) is a small protein involved in growth, maintenance and survival of some neurons. It binds to the receptor protein tyro sine kinase (Trk) which then activates multiple pathways depending on whether NGF is actin g on a cell that is developing or for cell survival. In developing cells it is known to a ctivate Ras guanine nucleotide-binding protein which activates downstream enzymes Raf and mitogen activated protein kinase (MAPK). When NGF and specific inhibitors of phospha tidylinositol-3 kinase (PI3K) were added to PC-12 cells, viability was reduced th rough apoptosis. The inhibition of Ras did not exert significant effects, and results indi cate and support the importance of PI3K to cell survival. Platelet derived growth factor (P DGF) was tested in place of NGF and yielded the same results (Yao and Cooper, 1995).
r Although Ras is predominantly part of the MAPK path way it does have some interaction with PI3K. Ras interacts with p85, a re gulatory subunit of PI3K, and causes activation of Akt in cell survival (Franke et al., 1995). In certain cases, such as in regulating sympathetic neuron survival, Ras is know n to initiate both MEK/MAPK (mitogen activated kinase kinase/ mitogen activated kinase) and PI3K/Akt. Akt activates TOR (target of rapamycin), a major factor that prom otes cell survival (Kennedy et al., 1997). The interactions are shown in Figure 2 below Figure2. The image shows activation of PI3K by direct interaction with the receptor as well as interactions with proteins such as Ras. Wortmannin, PI3K inhibitor, results in cell death as Akt is not phosphorylated and so cannot activate cell survival proteins such as S6-kinase (Kennedy et al., 1997). PI3K is a family of proteins that phosphorylate pho sphatidylinositol to phosphatidylinositol 3-phosphate (PIP), phosphatidy linositol 3, 4-bisphosphate (PIP2), and phosphatidylinositol 3, 4, 5-triphosphate (PIP3 ). Production of PIP2 and PIP3 attracts Akt to the cell membrane which binds to them throug h its Pleckstrin homology (PH) domain, a NH2-terminal domain which is widely found in enzymes t hat bind to
phosphatidylinositols. This site contains anti-para llel -strands and an -helix at the Cterminal (Cohen et al., 1995). Pyruvate dehydrogena se kinase -1 (PDK-1), a serine/threonine kinase is another kinase that has a PH domain, and is recruited by PIP2 and PIP3 to the cell membrane. Activation of Akt oc curs through PDK-1 phosphorylation of Akt at threonine 308 while another kinase, possi bly mTOR, phosphorylates serine 473 at the cell membrane (Leevers et al., 1999; Sun et al., 2005). The activation of Akt via phosphorylated phosphatidylinositol is illustrated in Figure 3 below Figure3. The figure shows PIP2 and PIP3 interaction s with the PH domain of PDK1 and Akt. PDK1 activates Akt (Leevers et al., 1999). The downstream effect of Akt does not include heigh tened expression of Bcl-2, a suppressor of cell death, compared to regular cellu lar levels. On the other hand, Bad, a member of the Bcl-2 family, and a pro-apoptotic fac tor is inactivated when phosphorylated at serine 136 by Akt. This was first seen through the activation by insulin growth factor I (IGF-1) which showed interesting re sults, namely the stimulation of Akt
without PI3K (Kulik and Weber, 1998). There was not iceable inhibition of pro-apoptotic Ced3/ ICE (interleukin1-converting enzyme)-like proteases which cleaves po ly (ADPribose) polymerase (PARP). Another downstream targe t of Akt is the inhibition of glycogen synthase kinase (GSK) which is another apo ptotic effector. GSK is thought to increase apoptosis by causing the breakdown of -catenin and therefore decreasing cell adhesion. TOR/FRAP (target of rapamycin/FKBP rapamy cin-associated protein) affects translation of proteins in the G1 stage and is pote ntially affected by Akt. This is likely to lead to some activation of p70S6-kinase which leads to the translation of proteins (Kennedy et al., 1997). The initial activation of Akt is reasonably well un derstood. Neurotrophins such as nerve growth factor bind to Trk receptors. Trk acti vates PI3K either by direct phosphorylation or it activates Ras and insulin rec eptor substrate-1 which can phosphorylate PI3K. The route of PI3K activation va ries depending on the type of neuron. PC12 cell (pheochromocytoma of rat adrenal medulla) survival is independent of Ras activation of PI3K, but this is not true of the superior cervical ganglion (SCG) or dorsal root ganglion (DRG) (Mazzoni et al., 1999). Stroke and trauma can occur in multiple areas of the brain. As a result, understan ding of the cellular pathway in each type of cell is likely to be important when promoting a therapeutic response. While many growth and survival factors initiate PI3 K, some do not. Camptothecin (CPT), a cytotoxic quinoline alkaloid, which inhibits DNA enzyme topoisomerase I, does not induce its response throu gh the PI3K pathway. Also, the activation of Akt is not solely through PI3K. Calci um/ calmodulin-dependent protein
kinase kinase is able to activate Akt resulting in phosphorylation of Bad (Yano et al., 1998). cAMP dependent protein kinase (PKA) is also able to activate Akt, although interestingly it does not act by phosphorylating Ak t. It is likely to inhibit phosphatases which would remove a phosphate from Akt and therefo re inactivate it. Activation of Akt through this pathway has been shown to inhibit GSK3 activity, although the method by which insulin and cAMP promote this is unusual as t hey would normally have opposites effect on GSK-3. Insulin would inhibit GSK-3 to pro duce glycogen, whereas cAMP would promote glycogen breakdown and therefore incr ease GSK-3 However, GSK-3 is known to be involved in more than glucose metabolis m, such as cell fate. Also the cell line was from a kidney which may not respond to GSK -3 the same way as liver cells (Filippa et al., 1999). In addition to Bad and GSK-3, a number of other do wnstream targets of Akt have been found. These include caspase 9, FKHRL1, L-Ca c hannel, IKK and eNOS (See Figure 4 below). In particular Bad, FKHRL1, L-Ca ch annel and GSK-3 are the main downstream targets in neurons, while GSK-3 is an essential component of the PI3K/AKT pathway (Hetman and Xia, 2000). Work on ox idative stress associated with Alzheimers disease demonstrated that IGF-1 activat ed PI3K induced production of NFB as an anti-apoptotic mechanism in neurons (Heck e t al., 1999).
Figure 4. A depiction of the factors that activate Akt and the proteins that it interacts with to promote cell survival (Hetman and Xia, 2000). As can be expected with the number of different fac tors involved in apoptosis mechanisms, there are elements in both the MAPK and PI3K pathways that interact with each other. In serum-deprived conditions, neuronal apoptosis caused the release of caspase-3. The addition of midkine, a neurotrophic factor from the heparin-binding family, activated both ERK and Akt. Inhibition of P I3K caused inhibition of Akt and extracellular signal-regulated kinase (ERK) showing that ERK has some impact on caspase-3 (Owada et al., 1999). Another investigat ion (Perkinton et al., 1999) found that Ca2+-permeable AMPA receptor activates MAPK/ERK via PI3 K. Ca2+-permeable AMPA receptors have an effect on cortical and striatal n eurons. This is remarkable as it included activation of a G-protein through an ionotropic rec eptor, AMPA. Cyclothiazide, a drug that stimulates AMPA receptors, was added to striat al neurons. This caused the dissociation of from the seven-transmembrane of Gi/Go-type G-proteins. moves to activate Ras via the PH domain of PI3K. Ras then st imulates the downstream pathway
MAPK/ERK. The activation of AMPA receptors also ini tiates cAMP response element binding (CREB) protein which activates gene transcr iption for short and long term neuroplasticity (Perkinton et al., 1999; Wang and D urkin, 1995)). These studies are important in investigating the cellular mechanisms associated with apoptosis and cell survival, but also how the various neuron types hav e different activated cellular signaling pathways. In this case the striatum neurons were sh own to use PI3K with MAPK. 9. MAPK Pathway Mitogen activated protein kinase (MAPK) includes a family of proteins that regulate many functions in all the cells of the bod y. The main MAPKs are extracellular signal-regulated kinase 1/2 (ERK 1 and ERK2), c-Jun amino-terminal kinases and p38 kinases (Figure 5 below). Activation of the cascade is similar to PI3K/Akt. Trk is activated by a neurotrophin and forms a complex wit h adaptor proteins, growth factor receptor-bound protein 2 (Grb2) and Sarcoma homolog y 2 domain containing transforming protein (Shc) through their SH2 domain s and phosphorylated tyrosine. Then a guanine nucleotide exchange factor Son of Sevenle ss (Sos) adds to the complex after which Ras is activated through changing a GDP for a GTP. Ras then activates many other proteins including the Raf family. Raf is a focal p oint of the whole pathway as many other factors act on it. This includes PKA, PKB/Akt and PKC. All the MAPK pathways use a similar mechanism of activation including MAP KKK, such as Raf, followed by MAPKK, such as MEK, and finally MAPK, such as ERK1/ 2 (Pearson et al., 2001).
Figure5. A diagram of mitogen activated protein kinase pathways. While p38 and JNK are generally pro-inflammatory, ERK1 and ERK2 (ERK ) can mediate an anti-inflammatory response (Roux and Blenis, 2004). The MAPKs are serine/threonine kinases and act thr ough a series of phosphorylations. This includes MAPK-activated prot ein kinases (MKs) such as ribosomal S6 kinases (RSK), MAPK-interacting kinase s (MNK), MAPK-activated protein kinases 2 and 3 (MK2 and 3) and MAPK-activa ted protein kinase 5 (MK5) (Roux and Blenis, 2004). Even within different cell types a certain pathway can have different effects. For example, (Ono and Han, 2000) p38 is kn own to be affected by factors such as platelet derived growth factor (PDGF), insulin like growth factor (IGF) and nerve growth factor (NGF). The functional significance of the ce ll type can be seen when insulin stimulates p38 in adipocytes, but down-regulates it in neurons. The cell type becomes more important as p38 is implicated in promoting ap optosis in neurons, but is blocked by both insulin and NGF (Kummer et al., 1997).
n The efficient interactions between the kinases and their substrates have been linked to docking sites. Docking-domains, positivel y charged residues surrounded by hydrophobic groups, can be found on substrates such as MK as well as scaffold proteins. These domains are important for the kinases to dire ct binding and phosphorylation of substrates (Enslen and Davis, 2001). Two research g roups also found a C-terminal common motif which is outside of the D-domain, and has acidic and hydrophobic residues. These residues interact with the D-domai n of proteins upstream and downstream of MAPK. Another domain which is importa nt for ERK 1/2 is the DEF domain (docking site for ERK and FXFP) which is uni que to ERK ability to phosphorylate its substrates. The domain includes p henylalanineX-aa (any amino acid) phenylalanineproline (Tanoue et al., 2000; Fantz et al., 2001) D domains play an important role in identifying wh ich cellular pathway, apoptotic or survival will be activated within a cell. When t he docking site of ERK-regulated MAPKAPK was changed to that of p38-regulated MAPKAP K (MK, a downstream protein), the result was a stress-activated kinase as opposed to a cell growth kinase. This shows the importance of docking sites in promoting specific cellular cascades (Smith et al., 2000). RSK1, a MK, was one of the first downstream effect ors of ERK found and has a D domain at the C-terminal. Since the discovery o f the RSK1, three additional human forms have been discovered, namely RSK 2, 3, and 4, and all four isomers have been shown to be present in the brain. Rsk 3 is expresse d at higher levels than the other forms, and is mainly found in developing neurons as well a s adult neurons of the cerebral cortex,
dentate gyrus, and amygdala. There is also some exp ression of Rsk2 in areas of the brain that have a lot more synaptic activity such as the neocortex, hippocampus, cerebellum, and Purkinje cell layer. Downstream effectors of Rs k2 in the hippocampus include CREB (Zeniou et al., 2002). CREB, which is phosphorylat ed at serine 133, is activated by Akt as well as Rsk, showing the close association of th ese survival pathways. Other substrates of RSK include the transcription factors c-Fos, c-J un, ER81 and NF-B. Rsk 1 and 2 promote cell survival through similar mechanisms as Akt, which phosphorylates Bad (Bonni et al., 1999), while Rsk1 promotes activatio n of NF-B through phosphorylation and therefore inhibition of IB (nuclear factor of kappa light polypeptide gene en hancer in B cells inhibitor, alpha) (Schouten et al., 1997 ). Another major family of downstream proteins for ER K 1/2 is the MSK proteins. MSK 1 and 2 phosphorylate CREB, but with higher aff inity than RSK1 (Deak et al., 1998). MSK 1 and 2 also interact with NF-B by phosphorylating serine 276 of p65 during stress (Vermeulen et al., 2003). They also p hosphorylate Bad and Akt as a result of UVB induced stress (Roux and Blenis, 2004). Oth er families of proteins such as MNK and MK may also interact with CREB and Akt. MK2 has a prevalent role in proinflammatory responses since of p38. MK2 knock-out mice had a smaller volume of stroke-affected cells and decreased expression of i nterleukin-1 (Wang et al, 2002). 10. MAPK+AKT in Stroke
The activation of MAPK/ERK and PI3K/Akt is known to be important in cell survival. Studies have illustrated that both respon d to the various neurotrophins through the Trk and p75 receptors, which inhibit caspases a nd Bad. An important study (Liot et al., 2004) used cortical neuron cultures to study M APK/ERK and PI3K/Akt, and there role in cortical neurons that are affected in strok e. NT-3, a neurotrophin, produces protective effects in adult neurons as opposed to t he effects of factors such as BDNF which are mainly influential in embryonic cells. Al though NT-3 seemed to use both pathways for the protective abilities, it was the A kt pathway that was more important for its anti-apoptotic effects. In this case, Akt was f ound to specifically target caspase 3, 8, and 9, and therefore decrease mitochondrial-breakdo wn induced apoptosis. i. Erk promoted cell survival: Erk has a very different role in stroke; promoting cell survival in certain instances, while promoting apoptosis in others. Studies have f ound a high level of phosphorylated Erk (pErk) in neurons and glial cells after stroke. In a study on the penumbra region of a stroke there was an increase in the amount of pErk and pCREB suggesting pErk has an anti-apoptotic role in this area. pErk was also act ive in the oligodendrocytes while another pathway, p38/SAPK, was active in the astroc ytes of this region (Irving et al., 2000). In PC12 cells, cells that mainly contain dopamine, the addition of NGF causes the cells to stop growing and to start differentiating. These cells were put into OGD conditions following eighteen hours of NGF pre-trea tment. The results showed an attenuation of p38 and JNK while pErk remained the same as that in the normal cell. On
r the other hand pErk was increased under normoxic co nditions (Tabakman et al., 2005). This suggests that MAPK/ERK is mainly active in cel ls as a neuroprotective mechanism as opposed to preventing apoptosis. This correlates with the high levels of Erk in the CA2-CA4 regions of the hippocampus, which has been resistant to ischemia (Sawe et al., 2008). Many studies found Akt to be a prevalent pathway in neuronal survival as well as Erk. However, some growth factors use Erk as the pr edominant pathway. Neuronal recovery in an in vivo study of hypoxic-ischemic brain injury occurred by BDNF activation of Erk, but not Akt (Han and Holtzman, 2 000). Another example of Erk protection is demonstrated t hrough the activation of Erk in the dentate gyrus and mossy fibers of the hippoc ampus after three minutes of exposure to ischemic conditions, as measured after two days. The cells were then re-exposed to ischemia, resulting in higher levels of pErk and de creased pJNK (Choi et al., 2006). ii. Erk induced apoptosis: Multiple studies have used Erk inhibitors to decre ased infarction. Wang et al. (2004) showed that the Erk pathway up-regulated int erleukin 1, although it had no effect on cytokines such as TNF. Studies in which neuroprotectants such as humanin result in smaller infarct size as a result of decreased Erk p hosophorylation (Sawe et al., 2008). Others focused on the impact of reactive oxygen spe cies (ROS) such as nitric oxide and peroxides. These species activate receptors such as epidermal growth factor receptor,
which starts a cascade with multiple MAP kinases. T his can lead to cell death especially when p38 and JNK are activated (Zhang et al., 2000) It has therefore been shown in multiple experiment s that the nature of the chemical niche at the site of injury can have an im pact on the outcome of the Erk pathway. It is clear that neuroprotective ERK 1/2 l asts around 24 hours in the penumbra after MCAO, and can be protective, while other type s of insult to the brain can lead to increased toxins that promote cell death through ER K (Chu et al., 2004). 11. Akt + MAPK + CB in stroke: A recent study focused on the influence of Akt and the MAPK pathways in disruption of the BBB. Akt is activated by ROS, VEG F and hypoxia induced BBB permeability. MAPK has been attributed to BBB openi ng in Alzheimers disease, TBI and focal ischemia. The opening of the BBB is thou ght to lead to some neuroprotection. Ultrasound was used to disturb the BBB by disruptin g the phosphorylated tight junction proteins occludin and ZO-1 (zona occluden). Of the proteins that were assayed, those central to the PI3K/Akt pathway were shown to be th e most elevated. Both pAkt and pGSK3 remained at elevated levels 24 hours after sonicat ion. The phosphorylated proteins are therefore found even after the BBB is closed. This suggests that VEGF causes Akt activation which then initiates neural s urvival (Jalali et al., 2010). Primary cortical neurons from embryonic day-18 rat s (rats with well developed cortexes) were cultured followed by testing with gl utamate-induced excitotoxicity
(Dasari et al., 2008). Initial results had a signif icant increase in the death of neurons cultured with glutamate compared with control. The addition of CB decreased the death of the neurons by half the number, although there w as still more death relative to neurons co-cultured with CB. Analysis of gene expression co nfirmed much of the information already known, namely the up-regulation of survival pathways, and the down-regulation both of stress pathways and the pathways that affec t NFB binding. The stress pathway was found to mainly induce caspase 3, and increase Bax levels, while that of Bcl-2 decreased. Therefore cell injury is, in part, cause d by ionotropic glutamate receptor activation. This was inhibited by an NMDA antagonis t and by CB. When looking at the effects of CB, Bcl-2 was increased leading to down regulation of caspases 3 and 7 and Bax. The final part of the study was devoted to the pathway that modulates the action of CB. The addition of Akt inhibitor IV caused signifi cant death even in the presence of CB. Western blot analysis demonstrated phosphorylated A kt was important in modulating CB effect against glutamate excitotoxicity. Another study by Lim et al. (2008) focused on the therapeutic effect of CB cells as mediated by both the ERK and the Akt pathways wh en activated by a growth factor such as BDNF. The researchers looked at the inducti on of CB-MSC to form neurons through treatment with neural induction medium. The cells were then added to the culture plates along with Akt and ERK inhibitors to determi ne their effects. The results showed BDNF activates both ERK and Akt, which are importan t for MSC differentiation to neurons and their survival. ERK increased the amoun t of Bcl-2 and p35, which has been implicated in neuroblastoma differentiation (Lee an d Kim, 2004). On the other hand, Akt increased the amount of Bcl-2 which would relate to its activation of the cell survival
pathway. Although both paths are necessary, Akt see ms to be the dominant anti-apoptotic pathway as it has been implicated in many regions o f the brain including the cortical and hippocampal regions. In this thesis study, we looked at the ability of CB to influence the MAPK/ERK and PI3K/Akt pathways in stroke like conditions. In contrast to the previous studies, we will use a transwell culture system that prevents d irect cell to cell interactions. The CB in this set of experiments will be placed on inserts ( view figure 6 in methods section below) which have 0.4um pores to allow any factors to move freely from CB to neurons and vice versa. This is therefore more likely to mimic the p resence of CB at the BBB and will give an insight into its response at the site of injury. It is my hypothesis that both Akt and ERK will have an influence in the protective abilit ies of CB, but the Akt pathway will be more significant.
Method: In order to address the question whether CB neuropr otection is mediated by Akt signaling pathways, we conducted a series of experiments in c ell culture combining the addition of CB cells to neuronal cultures and inhibition of Akt and MAPK signaling pathways. 1. Akt inhibitor Primary Neuron Culture: Female timed pregnant (embryonic day 17) Sprague-Da wley rats (Harlan) were anesthetized and then the pups h arvested and decapitated. The brains were removed and placed on ice in cold Dulbeccos m odified Eagles medium (Invitrogen, GIBCO DMEM media). The tissue was diss ociated and centrifuged. The cells were then counted and cultured in Neurobasal (Invitogen #21003-049) media supplemented with B-27 for six days. Media was chan ged on the second day and then every two days. Wells were plated at 1.5 x105cells/cm2. HUCB Cell Preparation: Cryopreserved umbilical cord blood mononuclear cell s were collected from AllCells LLC. The cells were thawed at room temperature and then washed twice in 10ml PBS/ 100uL DNase (Sigma # D452 7-40KU, 1mg/ml). Cells were stained with trypan blue, and counted using a hemoc ytometer with cell viability at a minimum of 80%. OGD Experiment: Invitrogen neurobasal A media (catalogue # 10888-022) was warmed at 37oC for 20 minutes and then flashed with 95%N2/5%CO2 gas for 20 minutes. Four conditions were set up, each with 4 ml of media. Th e 3 wells requiring CB required an insert with 1.5 x106 mononuclear cells added to the insert. To some wel ls 1.67uL/ml Akt
inhibitor IV (Calbiochem 124011) was added to the w ells. Media with inhibitors (4ml) was evenly split in the well and the insert. The cu lture plate was placed in an airtight hypoxic chamber (Billups-Rothernberg), flashed with 95%N2/5%CO2 for 10 minutes and then sealed tight for 20hours at 37oC in the Thermo water-jacketed CO2 incubator at 37oC 0.04%CO2. Between two and four replicates of the experimen t were performed. Control Experiment: The control conditions were set up as with the OGD experiment with Invitrogen neurobasal media (catalogue # 21103-049), but without flashing. Staining: To examine cell survival we used the fluorescein di acetate/ propidium iodide (FDA/PI) assay. We combined 5uL of 5mg/ml FDA in a cetone was added to 1ml 1xPBS to make the working solution. PI had a concentratio n of 1mg/ml in PBS. FDA (8uL/ml) and PI (6uL/ml) were added to 1x PBS, 1ml was added to each well. Fluorescent images were taken under the microscope. Cell Counts: Five pictures were taken of areas that were represe ntative of the general state of the cells in each condition. Live and dea d cells were manually tagged. Coaching included 10 hours of manual tagging to accurately d ecipher live cells from neurites (axons and dendrites). The number of live and dead cells in each picture was tabulated and used to find cell viability (live/total number of cells per image). There were four replicates of the Akti experiment. The five pictur es from each condition were counted, averaged and pooled with data from all replicates. This data was averaged and presented as mean + sem. An Analysis of Variance with Newman-Keuls po st-hoc tests was performed with minimum significance set at p < 0.05
2. MAPK inhibitor In this experiment we used 1uL/ml MAPK inhibitor (C ell Signaling Technology 9903) as well as 1.67uL/ml AKT inhibitor IV (Calbiochem 1240 11) in separate wells instead of the Akti. The same experimental procedures were pe rformed. In this case only two replicates were performed. The data is presented a s mean + sem. Since the data were not normally distributed, the data were analyzed with a Kruskal-Wallis test and a Dunns Multiple Comparisons test used for post-hoc analysi s. Figure6. An image of a 6 well plate and insert that was used to hold CB.
n Results: Akt study: In the first study, we examined cell survival of ne urons grown under normoxia or OGD conditions with or without CB, with or without Akti using the FDA/PI viability assay. FDA labeled cells (green) are alive while P I labeled cells (red) are dead. In both normoxia and OGD conditions, Akt inhibitor (Akti) i nduced widespread cell death (see Figure 7). Addition of CB to the cultures reduced cell death in the cultures with or without Akti added to the media.
Figure 7. Fluorescent pictures of the FDA/PI-labele d cells in normoxia conditions were taken at 20X. A) There was little cell death in neurons maintain ed in normoxia conditions as shown by the extensive FDA (green) labeled cells and few PI (red ) labeled cells. B) In cultures treated with CB, there were fewer PI labeled cells. The FDA labeled neurons fluoresces brightly with many neurites emanating from the cell body. C) In Akti treated c ultures, there is more cell death and the neurons have fewer neurites. D) In normoxia cultures trea ted with Akti and CB, there appears to be fewer PI labeled dead cells than in the Akti-treated culture s. Cell viability was determined by counting the numbe r of live and the total number of cells (live + dead) and then calculating a perce ntage. The data was analyzed with analysis of variance followed by post-hoc Newman-Ke uls comparisons. When we
counted the number of living and dead cells in each of the different groups, viability under normoxic conditions alone was 77.4 + 2.98 % (see Figure 8). When CB was added to the culture, viability increased to 89.9 + 3.3 % but this was not significantly different from the normoxia alone condition. Addition of Ak ti to normoxic condition significantly decreased viability to 47.5 + 11.72 % (p < 0.01). Viability in the normoxic + A kti + CB cultures was also significantly less than observed in the normoxia only condition (53.2 + 7.4 %, p < 0.05). Figure 8. Cell viability under normoxic conditions Cell viability between the four groups in normoxic condition (N) differed significantly (p<0.0001). Bar graphs are t he means of four sets of experiments (20 data points per condition, n=20) standard error of the mean (SEM). N vs. N+ Akt shows a significant de crease from 77.4 2.98 to 47.2 11.56. N+CB vs. N+Akti+CB also showed a significant decrease ( p<0.01) from 89.83.30 to 53.27.40 In contrast to the observations under normoxic cond itions, during OGD, a stroke-like condition in the culture dish, there wa s more cell death as shown by increased PI labeling of cells (Figure 9). There were still neurites emerging from the neurons in
r culture under OGD conditions, but there appeared to be more neurites when the neurons were exposed to CB during OGD. When Akti was added to the cultures, cell survival decreased and the neurons lost all neurotic outgrow ths, even in the presence of CB cells. Figure 9. Cells exposed to oxygen and glucose depri ved condition for 20 hours. Pictures were taken at 20X. A) The OGD conditions itself showed a mixture of cell death and live cells with fewer neurites. B) There were more live cells with neuritic outgrowth when CB was added to the culture. C) A large amount of cell death was observ ed when Akti was added to the cultures. D) Akti produced a large amount of cell death, but CB had some positive effects on the cells.
Cell survival in these cultures was calculated as d escribed for the normoxic conditions. In the OGD condition, cell survival wa s 43.9 + 7.7 % (Figure 10). After addition of the CB cells to the cultures, cell surv ival significantly increased to 79.0 + 6.3 % (p < 0.01). Cell survival in the Akti condition was 35.5 + 6.9 % and 46.7 + 2.2 % in the Akti + CB condition. These values were not sig nificantly different from the OGD condition, but were significantly less than the OGD + CB condition (p < 0.01). Figure 10. Cell viability under OGD conditions. Cell viability between the four groups in oxygen an d glucose deprived condition (OGD) differed significantly (p< 0.0001). Bar graphs are the means of four experiments (20 data p oints per condition, n=20) standard error of the mean (SEM) OGD+CB vs. OGD+ Akt+CB shows a significant decrease in cell vi ability (p<0.01) from 79.0 7.26 to 46.7 2.51.
MAPK study: The inhibition of MAPK/ERK had very interesting res ults. Just from observing the images of live and dead cells, it is clear that MAP K had little effect while the neurons were in favorable conditions (see figure 11). All t he figures show a healthy population of neurons, in normoxia conditions, many of which are congregated together. The amount of cell death in each image is clearly very small with the cells treated with just CB having the smallest number of dead cells. The inhibition o f MAPK had little effect on the neurons, as many of the axons can be seen and the c ells are all a regular size as opposed to being swollen or having parts of it budding off. When MAPKi was added to the culture, there was no change in cell survival (Figu re 11C. Adding CB to the cultures MAPKi also had little effect. Cell viability was calculated as with the previous experiment. The data were not normally distributed and so a Kruskal-Wallis test w as performed to analyze the data. Dunns Multiple Comparison Tests were done for post -hoc analysis. Although there were significant differences in the overall analysi s, (p<0.0001), there were no significance differences when comparing individual experimental groups in normoxic conditions (see Table 1).
Figure 11 Cells exposed to oxygen and glucose deprived condit ion for 20 hours. Pictures were taken at 20X. Live cells stained green with FD A. Dead cells stained red with PI. A) Neurons are aggregated in clumps with some dead cells. B) N eurons and CB with neurons grouped together. Image has brightly fluorescing neurons wi th visible axonal projections. C) Neurons in normoxia condition with MAPK inhibitor (MAPKi). Man y neurons with their axonal projections with dead cells intermingled through the image. D) Neurons with MAPK inhibitor and CB. Many neurons and axonal projections can be seen with few dead cells.
Table 1 Cell Viability in normoxic (N) condition with MAPK Inhibition Percentage viability was calculated as the average of 10 counts standard error of the mean (SEM). We also examined the effect of MAPK inhibition unde r OGD conditions. When the cultures underwent OGD alone, cell viability de creased as determined with FDA/PI (Figure 12). Addition of CB increased viability t o the level observed under normoxic conditions. When MAPKi was added to the cultures, there was no change in viability from OGD alone. However, CB cells could not revers e the OGD effects when MAPKi was present in the culture medium. When cell viability was calculated as described abo ve, viability for OGD condition was 53.1 3.2. The addition of CB increa sed the cell viability significantly (p<0.05) to 81.0 1.9. Cell viability in the MAPKi treated culture was 57.1 2.2; this was not significantly different from OGD alone. Ce ll survival in the OGD+CB+MAPK condition was 54.2 1.7 and was significantly lower (p<0.05) than cells in OGD+CB condition but not different from OGD alone or OGD + MAPKi. Data can be seen in Table 2. nr n r
Figure 12 Cells exposed to oxygen and glucose deprived condit ion for 20 hours. Pictures were taken at 20X. Live cells are stained green wit h FDA. Dead cells are stained red with PI. A) Neurons in OGD conditions with many cells alive but many of the axons receded. B) Few cells are dead, while many of the neurons show axonal pro jections. C) Inhibition of MAPK has resulted in considerable cell death, although many cells are still alive. D) Many cells are still dead, but CB has had some effect in cell survival.
Table 2 Cell Viability in oxygen and glucose deprived (OGD) condition with MAPK Inhibition Percentage viability was calculated as the average of 10 counts standard error of the mean (SEM). OGD and OGD+MAPKi+CB had significantly (p<0. 05) lower cell viability than OGD+CB. nr n n r n
n Discussion In this study, we examined whether the ability of h uman umbilical cord blood cells (CB) neuroprotective response to hypoxic and ischemic insults, such as oxygen glucose deprivation, is mediated by the Akt intrace llular signaling pathway, which is important in cell survival. In order to do that, w e cultured neurons in the presence of CB and exposed these cells to normoxic or OGD conditio ns. In some cultures we added Akt inhibitor (Akti) and in others we added MAPK inhibi tors (MAPKi). Inhibiting Akt led to extensive neuronal death both on its own and in the presence of CB. Cell viability dropped significantly (p<0.01) in normoxic conditio n from 77.4 2.98 to 47.2 11.56 when Akti was added. This shows that Akt is crucial to cell survival in favorable conditions. Comparing figure 7 A) and C) we see wid espread cell death in C) where Akti was added. While some cells are alive, they are swo llen and going through initial stages of apoptosis. The rest of the image includes axonal and dendritic projections which are beginning to break down. CB decreased in its neuroprotective effect when Akt i was present in the culture medium. In normoxic condition the addition of Akti to CB decreased cell viability significantly (p<0.01) from 89.8 3.30 to 53.2 7.4 0. In OGD conditions cell viability decreased significantly (p<0.01) from 79.0 7.26 to 46.7 2.51 with addition of Akti to CB. The substantial decrease in cell viability indi cates Akt has an important role in both normoxia and OGD conditions, and is a major pathway that mediates CB protective abilities.
In contrast to Akt inhibition, MAPK had no signific ant effect on neuronal survival in normoxic conditions. The images in Figure 11 in clude healthy cells that stain strongly with FDA. Comparisons between images C) and D) have little difference in cell viability which may suggest that CB has little effect in the presence of MAPKi. However, as there is very little cell death it is likely there was li ttle release of neuroprotective factors such as nerve growth factor, and therefore little stimul ation of CB protective abilities. The lack of significance when doing comparisons between expe riments in normoxia condition suggests MAPK is inactive when the cell is in favor able conditions. On the other hand, in OGD condition, the greater ce ll death in Figure 12 D) compared to B) means MAPKi decreases CB protective abilities. In B) axonal projections can be seen, but in D) there is little visibility of the neurites. This data indicates there was a clear difference in the cell viability of the neurons with OGD and OGD+CB+MAPK having significantly less (p<0.05) cell survival than OGD+CB. Therefore, this study indicates, in OGD conditions MAPK/ERK does become activated, and is important for CB to provide neuroprotection. When comparing the images for Akti and MAPKi, in OG D conditions, it seems as though Akti causes more cell death. However, there was a problem with staining. While the pictures with Akti showed a broad number of cel ls, those with MAPKi generally had very patchy areas. When comparing the pictures that included MAPKi to those of the neurons only there is a definite difference in the intensity of the green and red colors. However, the average cell viability had a low stand ard error of the mean suggesting the counts were replicated. A problem with this is that only two experiments were performed
with MAPKi. Even so, the fact that there was less c ell death when MAPKi was added suggests this pathway is not as significant as that of Akt in neurons. The experimental process certainly had problems as the media needed to be flashed and then separated so that the inhibitors c ould be added. Therefore the solution is likely to have been oxygenated by the time it was a dded to the wells and flashed in the chamber. Flashing in the chamber had problems as th e tubes needed to be clamped rapidly after shutting off the pure CO2 supply. If not properly clamped oxygen rapidly moves in leading to experimental failure. Also Akt i is light sensitive, and while I turned off the hood lights, surrounding light may have had some effect on the Akti during transfer. However, the experiments were replicated as much as possible. Other issues include the cell viability of the CB. Although tryp sin blue exclusion method was used to count the number of live cells there were some issu es with viability. Generally thawed fractions of CB have viability of 80-90%. However, after the first thaw the cell viability can decrease to 60%. Therefore using CB that contai ned many dead cells may have impacted its therapeutic response. The experiment demonstrates the influence of CB on the survival of the neurons and the importance of the two cellular pathways. CB is likely to have its effect through the release of factors, such as NT-3, NGF and BDNF which were able to pass through the semi-permeable insert. CB induced survival of neuro ns suggests it is able to have its effect at the BBB, as opposed to needing to have co ntact with the cells. However, BBB, which opens after injury, would need to be more per meable than in normal conditions for enough of the factors to go through (Belayev et al. 1996).
r The ability of CB to provide a therapeutic res ponse outside of the BBB suggests many therapies that can be used to target stroke as well as other forms of hypoxic conditions. A recent paper describes the im portance of CD 14+ monocytes and macrophages, as well as CD 133+ stem cells in CB (Sanberg et al. 2011). This confi rms earlier work that demonstrated mesenchymal stem cel ls are able to use the monocytes to suppress the proliferation of T-cells and therefore mediate the impact of mitogens (Culter et al. 2010). However, even with the addition of CB there was a s pecific time point at which it had its optimum effect. This is a problem that has resulted in failures for many other drugs, as many require treatment within 1 hour. As a result of the significance of time, a further experiment is needed in terms of looking at these pathways at times when the BBB is thought to first start opening. Although CB seems to work best at 48 hours, determination of the way in which inhibition of the pathways affects umbilical cord blood and cell viability at this point could help decide upon the relative impact of each cell survival pathway. Therapeutic effects could be base d on these factors including the upregulation of proteins increase efficacy of umbilic al cord blood. Li et al. (2003) demonstrated that Akt and ERK mech anisms are crucial in cell survival. At 1 hour post stroke pAkt is present in the central ischemic region while pERK is only found in the periphery along with pAkt. At three hours pAkt is absent in the central region, but it is in the penumbra along wit h pERK which peaks in amount at this time. Total amounts of both ERK and Akt had decreas ed by 24 hours. At all points however, more Akt was present relative to ERK. Ther efore relative to ERK, Akt has an
important role in cell survival and has greater inv olvement in the core region. The core goes through necrosis which results in rapid cell d eath which pAkt is unable to prevent. Following the presence of ERK in the penumbra for 2 4 hours, and therapeutic actions of CB after 24 and 48 hours, ERK is likely to have a r ole in cell survival as opposed to promoting apoptosis. Therefore looking at the potential use of CB at 4 h ours and seeing if it can increase the amount of pAkt and pERK at this time p oint could be extremely important especially if there are parts of the core that can still be saved. In conclusion, both the Akt and MAPK/ERK pathways a re clearly crucial to the survival of the cell in strokes and are also import ant to the therapeutic mode of action by CB. Akt is important for cell survival in normoxic and OGD conditions, and thus is likely to be constantly active as a cell survival pathway. On the other hand, ERK does not have a significant role in normoxic condition, but in OG D conditions at 20 hours it provides a mechanism for CB induced cell survival.
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