Le rôle du système immunitaire sur la
modulation de la neurogènese suite à un
accident aigu ou une maladie
neurodégénérative chronique
ThèseSENTHIL KRISHNASAMY
Doctorat en neurobiologie
Philosophiae doctor (Ph. D.)
Québec, Canada
© Senthil Krishnasamy, 2017
Le rôle du Système immunitaire sur la
modulation de la neurogène Suite à un accident
aigu ou une maladie neurodégénérative
chronique
Thèse
SENTHIL KRISHNASAMY
Sous la direction de :
Jasna Kriz, directrice de recherche
Résumé
L’interaction entre les cellules souches neuronales et les cellules du système immunitaire lors d’une condition pathologique n’est pas bien caractérisée. D’ailleurs, la communauté scientifique s’interroge sur l’implication des cellules du système immunitaire inné sur la régénérescence du système nerveux central à la suite d’un accident aigu ou d’une maladie neurodégénérative. L’objectif de cette thèse est d’investiguer l’implication d’un récepteur de type Toll, plus précisément le récepteur TLR2, sur la modulation de la neurogenèse lors d’un accident aigu et dans le cadre d’une maladie neurodégénérative.
Les récepteurs de type Toll font partie du groupe de récepteurs de reconnaissance de motifs moléculaires (PRR), activés à la suite d’une infection ou d’une blessure. D’ailleurs après une ischémie cérébrale, on peut observer par bioimagerie in-vivo une forte induction du récepteur TLR2. L’expression du récepteur de type TLR2 dans les neurones est principalement associée à deux régions : la région sous-ventriculaire du cerveau antérieur (SVZ) et la région sous-granulaire de l’hippocampe (SGZ). Nestin, une protéine impliquée dans la formation des filaments intermédiaires, est aussi fortement exprimée dans cette région par les cellules souches neuronales. C’est pourquoi nestin est utilisé comme marqueur de neurogenèse.
Pour le premier chapitre de ma thèse, nous avons généré un nouveau modèle murin afin d’étudier la neurogenèse avec l’imagerie par bioluminescence. En effet, la souris Nestin-luc/GFP s’avère un excellent modèle qui permet de suivre l’expression de nestin dans les cellules progénitrices neuronales (NPCs) après une ischémie cérébrale en temps réelle. Une forte expression de nestin fut observée par imagerie à 1, 3 et 7 jours suivant l’ischémie cérébrale. Pour le deuxième chapitre de ma thèse, nous avons estimé la population des cellules progénitrices neuronales chez les souris TLR2 KO à la suite d’une ischémie cérébrale. Chez ces souris, nous avons pu observer par immunofluorescence une baisse significative du nombre de cellules progénitrices
ischémie cérébrale. Pour le troisième chapitre de ma thèse, nous avons caractérisé un modèle murin de démence fronto-temporale (FTLD), la souris TDP-43A315T. Afin de
mieux comprendre le rôle du récepteur TLR2 lors de la neurodégénérescence nous avons croisé la souris TLR2-luc/GFP à la souris TDP-43A315T. Dans ce modèle murin
de FTLD, les symptômes cognitifs apparaissent vers l’âge de 7 à 8 mois et sont associés à une forte activation du récepteur TLR2. De plus, toujours grâce à l’imagerie in-vivo effectuée durant les 36 premiers mois de leur vie, on a pu observer des niveaux supérieurs d’activation du récepteur TLR2 durant la phase pré-symptomatique de la maladie comparée à la phase post-pré-symptomatique. Par la suite, à l’aide de marqueurs de neurogenèse (Nestin, DCX et Ki-67) un nombre de NPCs plus élevé fut observé par immunofluorescence chez les souris plus jeunes ce qui suggère une corrélation entre le niveau d’activation du récepteur TLR2 et la neurogenèse. En se basant sur ces évidences, je peux conclure que ma thèse a démontré l’importance de la voie signalétique du récepteur TLR2 après un accident cérébrale aigu et lors de troubles neurodégénératifs.
Abstract
Interaction between neural progenitors and immune cells under pathological conditions is not well understood. Still now researchers debate role of innate immune cells in the regeneration of central nervous system following acute injury and neurodegeneration.
Our aim is to investigate the role of Toll-like receptors in the modulation of neurogenesis followed by acute and chronic neurodegeneration.
Nestin, an intermediate filament protein, is expressed by neural stem and progenitor cells. Nestin is considered as a potential marker for neurogenesis. The two neurogenic niches of the brain, sub ventricular zone (SVZ) and sub granular zone (SGZ) produce neural stem cells throught the life. We have characterized transgenic mice to study neurogenesis by in vivo bioluminescence imaging. Our mouse model Nestin-luc/GFP mouse is an excellent tool to track the neural progenitors expression after stroke. In vivo bioluminescence imaging revealed that robust expression of nestin signal at 1 day, 3 days and 7 days following stroke.
Toll like receptors are the member of pattern recognition receptors, they activated upon infection and injury. In recent years, expression of toll like receptors in neuronal cells is well established in the two neurogenic regions of the brain. Activation of TLR-2 signal has been observed previously by in vivo imaging followed by stroke. Stroke induced neurogenesis is well characterised in these two neurogenic regions. Secondly, we have studied the role of TLRs in the modulation of neurogenesis followed by ischemia. In the TLR2KO transgenic mice, following cerebral ischemia, neural progenitors population were declined in both neurogenic regions compared to the wild type mice indicating TLR2 is necessary for the survival of neural progenitors in the ischemic brain.
TDP-43A315T is a transgenic mouse is valid mouse model of Fronto temporal
dementia. The onset of symptom will start at the age of 7-8 months. In this mouse, we have seen the higher expression of TLR2 in comparison to the wild type mice.
43A315T mice mouse model, TLR-2 expression was studied by in vivo imaging from
3months to 36 months. Longitudinal in vivo imaging showed an elevation of TLR2 signal in the pre-symptomatic phase of disease when compared to the post symptomatic transgenic mice. Next we have done an immunofluorescence analysis to estimate the NPGs population in the young and old mice. Up regulation of neurogenesis markers such as nestin, DCX and Ki-67 were observed in the young transgenic mice compared to the old mice which suggesting that TLR2 mediates the modulation of neurogenesis.
In this thesis work first, we have developed a mouse model to study neurogenesis by in vivo imaging. Secondly we have showed the importance of TLR2 in the regulation of neurogenesis after ischemia induction. Finally, mediation of TLR2 in the neurodegeneration and neurogenesis was observed. Our results showed the importance of TLR2 signalling in the brain following acute and chronic neurodegenerative conditions.
Table of Contents
Résumé ... iii
Abstract ... v
Table of Contents ... vii
List of Tables ... xv
List of Figures ... xvii
Abbrevations ... xix
Acknowledgements ... xxiii
Foreword ... xxv
Chapter 1: Introduction ... 1
1.1 Timeline of neurogenesis ... 2
1.1.1. Adult neurogenesis in the rodent hippocampus ... 4
1.1.1.1 Importance of adult hippocampal neurogenesis ... 5
1.1.1.3 Subgaranualr Zone (SGZ) ... 7
1.1.2 In vivo imaging of neurogenesis ... 9
1.1.2.1 Bioluminescence Imaging ... 9
1.1.2.2 In vivo imaging strategy ... 11
1.1.3 Neurogenesis in ageing ... 12
1.1.3.1. Adult neurogenesis in Parkinson’s disease ... 14
1.1.3.2 Seizure induced neurogenesis ... 14
1.1.3.3 Stroke induced neurogenesis ... 15
1.1.3.4 Neurogenesis after Traumatic Brain Injury ... 16
1.1.3.5 Neurogenesis in Huntingtons disease ... 17
1.1.3.6 Neurogenesis in Alzheimer’s disease ... 17
1.1.3.7 Alterations in neruogeneis in HIV associated cognitive disorders ... 18
1.1.4. Animal models of neurogenesis ... 18
1.1.4.1 Fluorescent markers for cell labeling ... 19
1.1.4.2 Nestin-based transgenic lines... 19
1.1.4.3 DCX-based transgenic lines ... 20
1.2 Inflammation in the nervous system... 21
1.2.1. Microglia ... 21
1.2.1.4 Microglia suppress destructive inflammation ... 24
1.2.1.5 The M1 and M2 microgla phenotypes ... 24
1.2.1.6 Microglial Involvement in Adult Neurogenesis ... 24
1.2.1.7 In vivo imaging of microglia in neurodegenration ... 26
1.2.2 Inflammation in stroke... 27
1.2.3 Neuroglial inflammation in ALS ... 31
1.2.3.1 Microglia in ALS ... 31
1.2.4 Cytokines and Neuroplasticity ... 31
1.2.4.1 IL-1α and IL-1β ... 33
1.2.4.2 IL-6 ... 33
1.2.4.3 IL-4, IL-10, and IL-11 ... 33
1.2.4.4 IFN-α and IFN-γ ... 33
1.2.4.5 TNF-α ... 33
1.3 Introduction to toll-like receptor (TLR) ... 34
1.3.1 TLR expression during development ... 34
1.3.2 TLR family... 35 1.3.2.1 TLR1, TLR2, AND TLR6 ... 36 1.3.2.2 TLR3 ... 36 1.3.2.3 TLR4 ... 37 1.3.2.4 TLR5 ... 37 1.3.2.5 TLR7 and TLR8 ... 37 1.3.2.6 TLR9 ... 37 1.3.2.7 TLR11 ... 37 1.3.3 TLR Signalling ... 38 1.3.3.1 TLR signalling pathways ... 39 1.3.3.2 MyD88-dependent pathway ... 40
1.3.4 Toll-like receptors mediation inflammation of the central nervous system ... 40
1.3.5 Toll like receptors in neurodegenerative conditions ... 43
1.4 Dementia ... 48
1.4.1. Frontotemporal lobar degeneration ... 49
1.4.2.1 Functions of TDP-43 ... 50
1.4.2.2 TDP-43 mutations ... 50
1.4.2.3 TDP-43 pathology in frontotemporal lobar degeneration ... 51
1.4.2.4 TDP-43 pathology found in other neurodegenerative disorders ... 52
1.4.2.5 Transgenic animal Models with TDP-43 pathology ... 52
1.4.3 Neuropathology of FTLD ... 53
1.4.3.1 FTLD-Tau ... 54
1.4.3.2 Frontotemporal dementia –Clinical aspect ... 54
1.4.3.3 Clinical features ... 55
1.4.3.4 Frontotemporal lobar degeneration-TDP ... 56
1.5 Hypothesis and Aims ... 58
Chapter 2: Molecular imaging of nestin in neuroinflammatory conditions reveals marked signal induction in activated microglia ... 63
2.1 Resume ... 64
2.2Abstract ... 65
2.3. Background ... 66
2.4. Methods ... 67
2.4.1. Experimental Ischemia ... 68
2.4.2. In Vivo Bioluminescence Imaging ... 68
2.4.3. Acute and chronic inflammation model (LPS induced inflammation): ... 69
2.4.4. Tissue Collection and Immunofluorescence ... 69
2.4.5. Primary cell cultures ... 70
2.4.6. Statistical analysis ... 71
2.5. Results ... 71
2.5.1. Cerebral ischemia is associated with strong induction of the nestin biophotonic/ bioluminescence signal ... 73
2.5.2. Acute and chronic LPS treatments induce robust and transient induction of the nestin-signals ... 75
2.5.3. Inflammation and brain ischemia increase microglial expression of nestin ... 76
Chapter 3: Acute inflammation in the TLR2KO mice affects the neurogenic niche .. 95 3.1 Resume ... 96 3.2 Abstract ... 97 3.3 Introduction ... 98 3.4 Methods ... 99 3.5 Results ... 101
3.5.1 Down regulation of neural progenitors in SVG ... 101
3.5.2 Down regulation of neural progenitors in DG ... 101
3.6 Discussion ... 102
3.7. References ... 105
Chapter 4: Early increase in inflammatory profiles and neurogenesis in mice with TDP-43 mutant mediated disease ... 107
4.1 Resume ... 108
4.2 Abstract ... 109
4.3 Introduction ... 110
4.4 Materials and Methods ... 111
Immunofluorescence ... 113
Cytokine array ... 114
4.5 Results ... 115
4.5.1 Early upregulation of TLR2 bioluminescence/biophotonic signals in TDP-43A315T transgenic mice ... 115
4.5.2 Exaggerated response to LPS challenge in pre-symptomatic TLR2-fluc-GFP; TDP-43A315T... 116
4.5.3 Increased Proliferation of NPGs in TDP-43 A315T mice ... 117
4.5.4 Ashwaghnada treatment promotes neurogenesis and improves the cognitive performance ... 118
4.6 Discussion ... 119
4.8 References ... 122
Chapter 5: General discussion and conclusion ... 133
5.1 Discussion ... 134
5.1.1 Characterization and validation of mouse model to study neurogenesis by in vivo imaging: ... 137
List of Tables
Table 1. Limitation of the Cre and Viral reporter lines. ... 20
Table 2 Effect of different cytokines on neurogenesis. ... 32
Table 3. Toll like receptor expression in neurodegenerative disorders. ... 44
List of Figures
Figure 1 Role of adult neurogenesis in the modulation of learning and memory . 3
Figure 2 Developmental stages in the hippocampal neurogenesis ... 5
Figure 3 Organization and lineage in the SVZ ... 6
Figure 4 Estimation of DCX+ cells in the GCL including SGZ across the entire human lifespan ... 9
Figure 5 Diagramtic representation of in vivo biophotonic/bioluminescence imaging from the brain of transgenic mice ... 11
Figure 6 Diagramtic representation of in vivo biophotonic/bioluminescence imaging from the brain of transgenic mice ... 12
Figure 7 Schematic representation of the adult hippocampal neurogenic events ... 25
Figure 8 Inflamamtion leads to secondary brain inflammation and cell death after ischemic injury ... 29
Figure 9 Cytokines in an adult brain and thier influence in neurogenesis ... 32
Figure 10 Expression patterns of TLRs and thier adaptor proteines throughout brain development ... 35
Figure 11 Structure of a human Toll-like receptor ... 36
Figure 12 TLR Signalling pathway ... 39
Figure 13 Cerebral ischemia induces Toll-like receptor signalling ... 41
Figure 14 TLR expression followed by ischem ... 42
Figure 15 Increased expression of TLR2 in the transgenic mouse model of FTLD ... 45
Figure 16 Decreased expression of DCX in TLR 2 KO mice ... 47
Figure 17 Dementia incresed in the numbers with advanced age compared to Parkinson ... 48
Figure 18 TDP-43mutations ... 50
Figure 22 Longitudinal imaging of nestin expression follwing transient focal ischemia ... 88 Figure 23 Expression of neural progenitors in SVZ, DG and stroke region ... 89 Figure 24 Series of in vivo imaging experiment followed by MCAO by acute and chronic inflammatory response in the brain of Nestin-luc/GFP mice ... 90 Figure 25 Increased number of GFAP and nestin positive cells after acute and chronic LPS administration ... 91 Figure 26 Iba-1 and Nestin expression pattern on brain after MCAO and LPSin Nestin-luc/GFP mice ... 92 Figure 27 Proliferative response of nestin+/Iba-1+ cells after inflammatory stimuli. ... 93 Figure 28 Expression of neural progenitors in WT and TLR2 KO mice after MCAO ... 103 Figure 29 Number of neural progenitors in WT and TLR2 KO mice after MCAO ... 104 Figure 30 Up regulation of TLR2 signal in FTLD mouse model ... 127 Figure 31 In vivo imaging of TLR2 activation following LPS challenge ... 128 Figure 32 Cytokine expression after LPS induction in the transgenic and WT mice ... 129 Figure 33 Neural progenitors expression in DG of both young and old WT and transgenic mice ... 130 Figure 34 Neural progenitors expression in VZ in both young and old WT and transgenci mice ... 131 Figure 35 Ashwagandha treatment induced the up regulation of neurogenesis in the FTLD mouse model ... 132 Figure 36 Cross - talk between TLR and PI3K signalling pathway ... 144
Abbrevations
AD: Alzheimer’s diseaseALS: Amyotrophic lateral sclerosis BDNF: Brain-derived neurotrophic factor BLI: Bioluminescence imaging
BrdU: Bromodeoxyuridine COX-2: Cyclooxygenase-2
DAMPs: Danger associated molecular patterns DCX: Doublecortin
DG: Dentate gyrus
DPP6: Dipeptidyl-peptidase 6
FALS: Familial amyotrophic lateral sclerosis
FTLD-U: Frontotemporal lobar degeneration with ubiquitinated inclusions FUS: Fused in sarcoma
EGF: Epidermal growth factor FGF: Fibroblast growth factor HD: Huntington disease Hsp70: Heat shock protein 70
hnRNA: Heterogeneous nuclear RNA
hnRNP: Heterogeneous nuclear ribonucleoprotein HVA: Higher vocal area
ICAM-1: Intracellular adhesions molecule-1
ICE: lnterleukin- -converting enzyme (caspase-1) ICV: lntracerebroventricular
IF: Intermediate filament
IGF-l: Insulin-like growth factor 1 IPC: Insoluble protein complexes
IκB: Inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells κ Kb: Kilobase
MAPKs: Mitogen-activated protein kinases MAPT: Microtubule-associated protein tau MCP-1: Monocyte chemotactic protein-1 M-CSF: Macrophage colony stimulating factor MCAO: Middle cerebral artery occlusion MHC: Major histocompatibility complex MN: Motor neuron
MnSOD: Manganese superoxide dismutase mTLE: mesial Temporal lobe epilepsy
MyD88: Myeloid differentiation primary response gene NF-L: Neurofilament light subunit (protein)
NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells NGF: Nerve growth factor
Nox-l: NADPH oxidase 1
Nrf2: Nuclear erythroid-2-related factor 2 OPTN: Optineurin
PAMPs: Pathogen associated molecular patterns PD: Parkinson disease
PDI: Protein disulphide isomerase PRR: Pattern recognition receptors RAG: Recombination activating genes
RANTES: Chemokine (C-C motif) ligand 5 (CCL-5)
RLD: Regulator of chromosome condensation 1 (RCC1)-like domain SALS: Sporadic amyotrophic lateral sclerosis
SGZ: sub granular zone
SLA: Sclerose laterale amyotrophique SOD l: Cu/Zn superoxide dismutase TAR: Transactive response element
TGF-β: Transforming growth factor beta TIR: Toll–interleukin-1 receptor
TRIF: TIR-domain-containing adapter-inducing interferon-β TLR: Toll-like receptor
TNFR: Tumor necrosis factor receptor TNF-α: Tumor necrosis factor-alpha
TRAM: TIR-domain-containing adaptor protein
TRIF: TIR-domain-containing adapter-inducing interferon-β UPS: Ubiquitin-proteasome system
VCAM: Vascular cell adhesion molecules VEGF: Vascular endothelial growth factor UBQLN2 : Ubiquilin 2
Acknowledgements
I would like to thank my supervisor Dr. Jasna Kriz for giving me an opportunity to come to Canada for my graduate studies. Her constant support and encouragement lead me to finish my thesis work. Without her inputs, it wouldn’t be impossible to accomplish my doctoral degree. Every scientific discussion with her enhanced my knowledge in the field of research and helped me to think and act independently. She is an independent and well renowned scientist in the field of neuroimmunology in world. I am very thankful for her support at the time of difficulties in the past five years.
I would also like to acknowledge Dr. Jean-Pierre Julien for his motivation and for allowing me to use his lab facilities. Participation in annual meetings held every year Andre-Delambre Annual Symposium conducted by Dr. Jasna and Dr. Jean Pierre Julien was very useful to enrich our knowledge and keep updated about ongoing research on ALS and other neurodegenerative diseases.
I would like to extend my special thanks to my doctoral committee members Dr. Armen Saghatelyan, Dr. Frédéric Calon, Dr. Emmanuel Planel for their thoughtful comments and suggestions. I want to thank neurobiology program director Dr. Katalin Toth for her valuable guidance.
My sincere thanks to Dr. Melanie-Lalancette Hebert, who gave lot of inputs for my thesis. Her critical comments and valuable suggestions improvised my work in a well-organized manner. She helped me lot in the preparation of my manuscripts. I want to thank Dr. Daniel for his help in transgenic mouse construction.
My grateful thanks to our wonderful research assistants Yuan Cheng Weng, Geneviève Soucy and Christine for their help in my experiments. Special thanks to my lab members Pierre, Mathieu, Sai Sampath, Hejer, Reza, Louis, Kallol, Prakash,
In the past five years, I had an opportunity to get nice friends from all over the globe. I would like thank all of them including Arojit Mitra, Christopher, Ranjan Maity, Ghazal, Hemanta Adikary, Anidita, Sharbani, Pranav kumar, Namarta, Preyesh, Jina, Dineshbabu, Prenitha Mercy, Ramesh Muddala, Naveen Kumar, Minty Thomas, Deepak kumar Jha, Likun Panda, Hemalatha, Vivek Mahadevan for their wonderful company.
Special thanks to my lovely sisters Mathivanai and Malarvzhi and My brother-in-laws Muruganandam and Subramani, nieces Sowmiya, Harsha and nephews Karthi, Sriram for their unconditional love and care.
Heart felt thanks to Gowsica Ramakrishnan for her unconditional support and care. She has given me a lot of confidence and support during my hard times, which helped me to focus in my research.
I dedicate this thesis to my parents.
Thanks to God for giving me strength and ablility to complete my thesis work.
Thank you Senthil
Foreword
This thesis includes a literature review that outlines the current knowledge and recent findings about neurogenesis, neuro-immune recations including microgila, Toll-like receptors (TLRs) and TDP-43 invlovement in Fronto temporo loabar demntia (FTLD). The first part of the first chapter discusses the importance of neurogenesis, animal models of neurogenesis and in vivo imaging of neurogenesis. The second part of the first chapter reveals the relationship between the immune cells and the neural stem cells. Third part of the first chapter deals mostly about basics of toll like receptors and their involvement in neuronal injury and neurodegeneration. The final part of the first chapter contains the clinical and pathological features of FTLD. The second chapter of this thesis is presented in the form of a research manuscript where I am the principal author. The manuscript submitted to Journal of Neuroinflammation:
“Molecular imaging of nestin in neuroinflammatory conditions reveals marked signal induction in activated microglia”. Senthil Krishnasamy, Yuan-Cheng
Weng, Daniel Phaneuf, Melanie Lalancette-Hebert and Jasna Kriz.
Third chapter of this thesis consists of the fate of neural progenitors in TLR2KO mice followed by transient focal ischemia. The manuscript is accepted for publication in Journal of Neuroinflammation
Fourth chapter will be submitted to the journal of Brain, Behaviour, and Immunity: “Early increase in inflammatory profiles and neurogenesis in mice with TDP-43
mutant mediated disease”. Senthil Krishnasamy, Sai Sampath Thammisetty,
Kallol Dutta, Melanie Lalancette-Hebert and Jasna Kriz.
1.1 Timeline of neurogenesis
Santiago Ramony Cajal believed that new neurons in the brain will form only during the pre-natal development. This concept of ‘no new neurons after birth changed after the identification of stem cells in the two regions of the brain sub ventricular zone and dentate gyrus (Ramon y Cajal et al., 1913).
Historically, the first report about neurogenesis came in the 1960’s by Altman and Gopal das in rodents. Thirty years later, three researchers made a remarkable discovery about neurogenesis by showing the formation of new neurons and their function and implication in adults (Gould & Gross, 2002).
Now there are many studies are going on in the field of adult neurogenesis in physiological and pathological conditions including Alzheimer’s disease and Parkinson’s disease. In addition to that some of the studies are showing the new neuronal cells contributes to recovery from trauma and stroke.
Several theories have been suggested for the relationship between increased neurogenesis and learning, memory or improved cognition; new neurons may increase memory capacity, reduce interference between memories and could be crucial for placing time details onto memories (Lafenetre et al., 2011). Figure.1
Figure 1 Role of adult neurogenesis in the modulation of learning and memory
Upreguation of adult neurogenesis (pink arrows), experience, enriched environment and physical activity (blue arrows) stimulate the generation of new neurons. This could thus lead for better performance in learning and memory tasks (Lafenetre et al., 2011)
1.1.1. Adult neurogenesis in the rodent hippocampus
Adult hippocampal neurogenesis in mammalian hippocampus involved in cognitive functions that is essential for humans. Adult neurogenesis is a multi step process; it is a single event.
Neurogenesis is a series of events that are all important to produce new neurons. Using immunohistochemical methods, the new neurons were marked by the presence of birth marker with the thymidine or BrdU method (Kuhn et al. 2015) a couple of weeks earlier. The expression of polysialilated neural-cell-adhesion molecule (PSA-NCAM) present in neurogenesis has been identified in early but that is not relevant to proliferation or the mature stage (Seki and Arai 1993a, b).
Adult neurogenesis has been divided into four phases: a precursor cell phase, an early survival phase, a postmitotic maturation phase, and gliala late survival phase. (Figure 2)
Figure 2 Developmental stages in the hippocampal neurogenesis
(Kempermann G et al., 2015)
1.1.1.1 Importance of adult hippocampal neurogenesis
Hippocampal neurogenesis plays an important role in learning and memory. Physical activities like exercises were shown to promote hippocampal neurogenesis resulting in improved memory formation (Van Praag et al., 1999). The link between hippocampal neurogenesis and antidepressants has also been identified. Treatment of antidepressants were proven to up regulate neurogenesis in the hippocampus (Malberg et al., 2000). The disruption of hippocampal neurogenesis seems to affect only certain types of memory. Studies have demonstrated that impairment was selectively seen in trace eye-blink conditioning and trace fear conditioning but not
On the other end is the implication of hippocampal neurogenesis in pathology. Seizure induced neurogenesis is thought to contribute to epiletogenesis and to long-term cognitive impairments (Jessberger et al., 2007).
1.1.1.2 Subventricular Zone (SVZ)
Lateral ventricles are the one of the main sources for stem cells in the brain. They give rise to neuroblasts that follows the rostral migratory stream to reach the olfactory bulb. Architecture of the human sub ventricular zone was investigated by Arturo Alveraz- Buylla and colleagues in 110 surgical and postmortem specimens (Sanai et al., 2004). In that study, lateral verntricles of the brain comprises a ribbon of glial fibrillary acidic protein (GFAP)-expressing astrocytes along the ependymal cell layer and that is different from the rodent sub ventricular zone. The interaction in rodents of migratory neuroblasts (type A), astrocytic stem cells (type B), transiently amplifying cells (type C), and ependymal cells was not seen in the human sub ventricular zone (Figure 3).
Cross section of the anterior rodent brain indicating the location of the SVZ on the lateral wall of the LV. On the right is the cellular composition and organization of the SVZ (Alvarez-Buylla and Garcı ́a-Verdugo et al., 2002).
Human fetal forebrain has a high proliferative activity with cells coexpressing the migrating neuroblast marker DCX and PSA-NCAM (Wang et al., 2011). Same kind of findings was reported in the infant forebrain, in which expression of DCX and the immature glial marker vimentin in the subventricular zone (Sanai et al., 2011). Neuroblasts either migrated tangentially or some of them formed chains within the rostral migratory stream and these chains were similiar with chains of migratory neuroblasts that are present in SVG and like that of rodent rostral migratory stream and monkey brain (Sanai et al., 2011; Wang et al., 2011). Infant forebrains of human consist a supplementary migratory stream of DCX (Doublecortin)-positive cells were identified. After birth the in the migratory strems proliferation and DCX expression will be reduced. Nearly at 6 to 8 months of age very few migratory neuroblasts and prolifrative cells can be seen in the rostral migratory stream (Sanai et al., 2011).
1.1.1.3 Subgaranualr Zone (SGZ)
In mice, hippocampal neurogenesis has been characterized and the markers for different developmental stage markers also identified. (Kempermann et al., 2004). To charectrize further, Kempermann and his colleagues studied the features of rodent hippocampal neurogenesis in human brain. In their study, they found out the the pattern of DCX-positive cells in the adult human DG, using many samples (n =55) ranging from the age from 1 day to 100 years (Knoth et al., 2010). Across the lifespan, DCX-positive cells were found in the DG of human brain (Figure 4).
Ki67 and Mem2, showed cell proliferation up to at least the ages 38 and 65 years, respectively.
Stereological studies conducted in the adult human brain revealed that neuronal cells decrease with the age, at the same time neuronal number in the DG are less affected. (West and Gundersen 1990; West 1993; Simic et al., 1997; Harding et al., 1998; Korbo et al., 2004). Among the newly generated neurons, death of young neurons happenes faster, this proves that preferential loss of adult-born neurons.
The neuronal exchange rate compared with adult humans of 0.004% DG per day can be compared with 0.03% to 0.06% per day in 2-month-old mice and 0.004% to0.02% per day in 5- to 16-years -old macaques (Kempermann et al., 1997; Kornack and Rakic 1999; Jabe`s et al., 2010). In mice, at 2 and 9 months of age, there is a 10-fold decline in neurogenesis (Ben Abdallah et al., 2010). These results indicate that adult hippocampal neurogenesis in middle aged mice can be compared with humans. .
Figure 4 Estimation of DCX+ cells in the GCL including SGZ across the entire human lifespan
(Knoth et al., 2010)
1.1.2 In vivo imaging of neurogenesis
Neruroimaging methodology is useful to detect various physiological and pathological effets on neurogenesis. Phsyiological activities like enriched environment and learning, physical activity, aging directly influence on neurogenesis. Effects of these activities modify the hippocampal volume that has been studied by various researchers using imaging methods.
of luciferase enzymes. This enzyme can produce light by oxidizing a substrate (i.e., D-luciferin) by utilizing molecular oxygen and energy. The photons emitted from the living tissues can be detected by a camera and quantified by highly sensitive charge coupled device thus allowing us to analyse the different aspects of biomoleular reactions in a wide range of living organisms including transgeneic mice.
Bioluminescence imaging has been given greater attention in biological research. Due to its simple and validated methodology, mechanisms of various disorders and its therapeautic effects were analyzed such as in cancer, other infectious diseases and neurological disorders.Bioluminescence operates as a biochemical reaction, so there is no exterenal resource requires to obatain a signal.The photons released by reporter mice constructed with luciferase pass the tissue and are able to capture at the surface with sensitive photo detectors based on a CCD camera (Kriz J et al., 2009) (Figure 5)
Figure 5 Diagramtic representation of in vivo biophotonic/bioluminescence imaging from the brain of transgenic mice
(Kriz.J et al., 2009).
1.1.2.2 In vivo imaging strategy
In 2012, Cordeau et al., reported an excellent explanation for the strategy behind the in vivo bioluminescence imaging. It is a pure chemical recation, which starts by prior to the imaging mice anaesthetized and injecting D-luciferin that induce chain of
next the luciferly adenylate under the influence of AMP prodcues luciferly dioxetanone and finally the oxyluciferin casues the photon emission which can be captured by CCD camera.
Figure 6 Diagramtic representation of in vivo biophotonic/bioluminescence imaging from the brain of transgenic mice
(Cordeau P et al., 2012)
1.1.3 Neurogenesis in ageing
There exists a negative relationship between aging and neurogenesis (Kuhn et al., 1996; Luo et al., 2006; Olariu et al., 2007). A reduction in neurogenesis with increasing age has been reported under laboratory conditions in rats (Kuhn et al., 1996; Olariu et al., 2007), canines (Siwak-Tapp et al., 2007), marmoset primates (Leuner et al., 2007), and in free-living animals (Barker et al., 2005). It has been reported that old DG neurogenesis is more than 80% less than in young adults (Kuhn et al., 1996; Cameron and McKay, 1999; Olariu et al., 2007). Although a gradual decrease in birth and differentiation of new GC is observed, most of the new cells in old rats are arranged in clusters along the SGZ, in the same pattern observed in the young ones (Kuhn et al., 1996).
hyperbolical, with a strong decline in the first years of life and a low, but persistent reduction in the rest of the life span (Barker et al., 2005). There is also a difference between free-living animals; in adult squirrels, there is a decrease in proliferating cells but not in new neurons and, on the contrary, in adult chipmunks, the decrease is in the density of young neurons. These differences might occur because of the use of distinct memories in the survival strategies of these species, for example squirrels use more spatial memory, because they have multiple food storage sites (Barker et al., 2005).
The process of decline in neurogenesis remains unclear. Different studies suggest that a loss of precursor cells or a slowing in the cell cycle progression may contribute to the reduction in neurogenesis in aging SGZ. Olariu et al.,2007 observed a decline in dividing precursors and no change in the duration of the cell cycle of the dividing cells in the DG. On the other hand, Luo et al. (2006) observed an age-dependent decrease in cells positive for BrdU, and a decrease in the ratio of BrdU-positive cells in S phase to the total number of cycling cells, as shown by the general S phase marker Ki67.
In the lateral ventricles, a thinning of the SVZ has been observed in aging compared to young and juvenile mice (Luo et al., 2006). Reason for thining of SVZ is due to a reduction in the number neurons and a decline in the birth of new neurons. Different groups have described that there is more than a 50% decrease in SVZ neurogenesis in old compared with young adult animals (Tropepe et al., 1997; Maslov et al., 2004; Luo et al., 2006). Luo et al., (2006) observed a reduction in cell proliferation; an increased number of astrocytes, and that neuroblasts and neurogenesis in general has become restricted to the anterior dorsolateral horn of the SVZ (Luo et al., 2006). This observation contradicts the findings of Kuhn et al., (1996) showing that the age-related attenuation of proliferation is specific for the SGZ.
al., 2006).
1.1.3.1. Adult neurogenesis in Parkinson’s disease
Parkinson disease (PD) is considered as a most prevelant neurological disorder in central nervous system. Pathogenesis of PD starts with reduction of neurons in the small midbrain structure substantia niagra. Examination of neurogenesis in Parkinson disease patient’s brains, showed that strong intense staining of nestin in the substantia niagra was observed (Yoshimi et al., 2005) at the same time many number of PSA-NCAM positive cells were detected in the SN pars reticulate in the PD patients compared to the normal subjects. Rats and macaque monkeys also had more number of PSA-NCAM positive cells than the normal intact side.
In 2004 Hoglinger &Baker showed a less number of NSC in the SVZ of PD models. Liu el al.2006 showed that an increased number of neural progenitors in the PD models by unilateral lesion to the nigrostriatal pathway by injection of 6-hydroxydopamine (6-OHDA). There is a difference in the occurrence of neurogenesis in acute and chronic models of PD. In the acute model, there is an increased number of GFAP cells and less number of PSA-NCAM positive cells in the ventricular zone. Dopamine is an important component in neurogenesis, therefore many researchers interested to look out the neurogenesis in PD. Neural stem cells should migrate and differentiate to work in the functional circuits. Cohen et al., 2004 claimed that functional recovery in the PD model due to the new born cells.
1.1.3.2 Seizure induced neurogenesis
The initiating site of seizures called as seizure focus in patients suffering from mesial temporal lobe epilepsy (mTLE), typically hippocampal formation and neighbouring structures of the mesial temporal lobe. In late 1990s, by series of experiments in the
after the Status Epilepticus (Parent et al., 2002). In the dentate gyrus after the several days of SE, increased number of DCX positive cells were found, suggesting that SE induced neural progenitors induction in the hippocampus (Jessberger et al., 2005). The number of dividing progenitor cells formed due to the SE returns to the control levels after 4 weeks of SE (Parent el al., 1997, Jessberger et al., 2007).
The molecular mechanisms behind the formation of new neurons after SE is might be due to the ‘electrical activity’ sensing capacity of NSC (Deisseroth et al., 2004). Another possibility is that the supportive role of transcription factors (BDNF, VEGF) in the surrounding structures (Newton et al., 2003).
The granule cells which form under normal condition in the hippocampus undergo specific maturation steps to acquire enhanced excitability. In contrary to that in the SE induced neurogenesis faster rate of maturation occur due to the seizure activity (Overstreet-Wadiche et al., 2006). Not only the faster maturation, in addition to that the newly formed granule cells extends abnormal dendritic processes towards the hilus (Shapiro et al., 2005, Ribak et al., 2006), and the spines from the dendrites also sprout dramatically and that contribute hyper excitability (Parent et al., 1999). So, SE induced neurogenesis produce new neurons and contributes to the aberrant growth of dendrites and spines of granule cells. Still it remains unclear that these newborn cells are helpful or detrimental to the brain. Seizure induced neurogenesis would be an interesting target to understand better strategies for the treatment of epilepsy.
1.1.3.3 Stroke induced neurogenesis
Occlusion of cerebral artery results in an irreversible damage to the brain. Until now there is no effective treatment is available for stroke. At the time of occlusion many different type of cells (glia, neuron etc) die in the brain. Identification of neural progenitors following stroke in the neurogenic regions open a new avenue for the treatment of stroke (Jin and Gavan et al., 2007, Zhao et al., 2008).
survive after 1-2 weeks of stroke (Jin et al., 2001, Arvidsson et al., 2002, Parent et al., 2002). This proves that stroke causes long term alteration in neurogenic niche of SVZ. The Molecular mechanisms underlying the proliferation of newborn cells in the SVZ are unknown. Notch 1 is the important factor that promotes neurogenesis, by showing infusion of Notch 1 produces more number of progenitors in the SVZ following stroke. IGF-1(Insulin like growth factor) is also considered to be involved in the neural progenitor proliferation in SVZ after stroke (Yan et al., 2007). TNF-R1 is the sub-type of TNF-α (Tumour necrosis factor) also influence the neurogenesis following stroke in SVZ (Iosif et al., 2008). Initially it is believed that neurogenesis following stroke is a short-term event, but in 2006 Kokaia and Thored showed that in the rats after 2hrs of MCAO formation of striatal neurons continue till 1 year. Migrating neuroblasts marker doublecortin (DCX) expressed in the penumbra region, they survive over 30 days after injury. Studies showed the possibility of stroke induced neurogenesis in human brain. Macas et al .2006 showed that more number of ki-67 positive cells in the ipsilateral SVZ and an increased number of neural progenitor cells along the wall of ventricles (Jin et al., 2006). Another important study conducted in 84-year-old stroke patient brain, has increased number of Nestin, Sox2, Musashi and PSA-NCAM in the region of Infarction (Minger et al., 2007).
1.1.3.4 Neurogenesis after Traumatic Brain Injury
Injury to brain activates neural progenitor cells in the SVZ and DG. These new born cells differentiate into glial cells in the injured cortex and finally integrate into the hippocampus as neurons. Neural progenitors in the SVZ have an ability to migrate to the injured area, in contrast hippocampal neurogenesis setting to diffuse injury. Collectively total number of new neurons produced after TBI are comparatively lesser than the astrocytes and oligodendrocytes. Using different type of TBI models researchers demonstrated that bilateral proliferation in the SVZ (Chirumamila et al.,
the induction of neurogenesis after TB1. Remarkable recovery from the TBI due to the contribution of neurogenesis effect.
.
1.1.3.5 Neurogenesis in Huntingtons disease
Many reports showed that neurogenesis occur in the animal models of HD and post-mortem brains from HD patients. Curtis et.al.2003 and 2005 observed that increased number of Proliferating Cell Nuclear Antigen (PCNA) in the SVZ of HD patient brains. In the double immunofluorescence studies 50% of the PCNA positive cells express GFAP (Glial fibrillary acidic protein) and 3% express in the neuronal marker β111- tubulin. PCNA is indicator of ongoing cell division and DNA repair (Curtis et.al.2003)
1.1.3.6 Neurogenesis in Alzheimer’s disease
In Alzheimer disease, widespread neuronal loss occurs in the cortex and limbic system and deposition of Aβ in the neuropil and formation of neurofibrillary tangles (Terry et al., 1994). It is well known that neurogenesis play a main role in memory and learning. Study conducted by Jin et al., 2004 b in post-mortem brains of AD patients showed an increased neurogenesis and it may be a compensatory mechanism to the neurodegeneration.
In the transgenic mouse models of AD there is a significant alteration in the hippocampal neurogenesis (Dong et al., 2004, Jin et al., 2004a, Wen et al., 2004). Rockenstien et al.2006 showed a decreased neurogenesis in SGZ of the DG, with decreased numbers of cells labeled with markers of neurogenesis such as DCX and BrdU and concomitant increase in the expression markers of apoptosis. In another mouse model combination of AD transgenic overexpression APP and PS-1 showed a marked reduction of neurogenesis (Zhang et al., 2007).
around senile plaques generating positive staining for proinflamatory mediators like cyclooxygenase 2 (Cox-2), interleukin-1 (IL-1) and IL-6, and elevated levels of cytokines and chemokines (Nelson et al., 2002; Liu and Hong, 2003; Glass et al., 2010), which at the same time, are harmful for the adult neurogenic process (Ekdahl et al., 2003, 2009; Das and Basu, 2008; Ghosal et al., 2010). Therefore, it is thought that anti-inflammatory therapy could be appropriate to restore the levels of neurogenesis in AD brains.
1.1.3.7 Alterations in neruogeneis in HIV associated cognitive disorders
HIV-infected macrophages enter CNS leads to inflammatory condition with astrogliosis, microglial cell activation, and myelin loss and synapatodendritic damage (Ellis et al., 2007). HIV-associated dementia symptoms reduced with Highly Active Antiretroviral Therapy (HAART), chronic forms of HIV Encephalitis (HIVE) with moderate neurological and psychiatric disturbances still exist. HIVE represents an important inflammatory condition that leads to cognitive impairment and neurodegeneration. To understand the alterations in neurogenesis in patiets with HIVE may helps to elucidate mechanisms through which inflammatory conditions lead to neurodegeneration and abnormalities in neurogenesis.
1.1.4. Animal models of neurogenesis
There are various strategies to visualize, identify, and enumerate stem cells and their progeny in the adult brain in vivo. Traditionally, studies of neurogenesis were relied on immunocytochemical staining of brain sections using stem-cell-specific antibodies and their combinations and on marking dividing stem cells and their progeny using thymidine analogs. These techniques are now complemented by powerful genetic approaches for ontogenetic labeling: generation of transgenic reporter animals constitutively expressing marker proteins; indelible labeling of stem cells and their
The available informations about adult neural progenitor cells and their progeny has been obtained using corresponding transgenic mouse lines with their genetic markers.
In such lines, a specific promoter, by directing expression of a Fluorescent Proteins (FP), helps to identify cells, their subpopulations, or defined classes of their progeny. The range of such lines is steadily expanding, providing an abundant choice of reagents to probe adult stem cells. This general strategy is increasingly supplemented using inducible transgenic mouse lines, in which Cre recombinase is activated by tamoxifen or doxycycline at a given time point to mark the progeny of the cells that have undergone recombination; Again, a steadily growing collection of inducible lines facilitates the choice of genetic reagents.
1.1.4.1 Fluorescent markers for cell labeling
A key component of constitutive, inducible, or viral reporters is an FP driven by appropriate regulatory elements. The palette of FP expression in transgenic reporter lines is constantly increasing, with GFP, cyan fluorescent protein (CFP), DsRed, mCherry, and tdTomato were routinely used. In addition, more advanced variants of FPs are continuously being generated and characterized for the purposes of genetic cell labeling.
1.1.4.2 Nestin-based transgenic lines
Nestin-GFP mice have been used to study the physiological properties of neural precursors and the signaling mechanisms involved in their maturation. Three FP lines that use the same promoter and enhancer elements derived from the rat nestin. In Nestin-GFP mice (Mignone et al., 2004), the fluorescent signal highlights all the soma and the processes of stem cells and early progenitor cells, and these mice are well suited for the studies of the distribution and morphology of neuronal precursors in developing and adult brain. In Nestin-GFP/ luc mice (Encinaset al., 2006) the signal is localized in the cell nucleus and the distribution of the stem and progenitor cells is visualized as a punctuate pattern. This nuclear localization of neural stem cells reduces the complexity of their expression pattern and allows their unambiguous
1.1.4.3 DCX-based transgenic lines
DCX is a microtubule-associated protein critically involved in neuronal migration and neurite outgrowth that is widely used as a marker for phenotypic identification of neuronal precursors and immature neurons. DCX is not expressed exclusively in newborn cells, as it can be re expressed in certain populations of mature neurons that are undergoing structural plasticity (Nacher et al., 2001). However, in the DG and olfactory system, it is reliably expressed in neuronal precursors and newborn neurons (Brown et al., 2003; Couillard-Despres et al., 2005). Couillard-Despres and his colleagues (2006) generated transgenic mice using the identified 3.5-kb fragment upstream of the DCX ATG start codon to drive expression of GFP or DsRed. Labeled cells have a variety of immature morphologies, consistent with the range of developmental stages that are characterized by DCX expression. DCX-based transgenic reporters are used to find immature cells in old mice and to isolate immature neurons for gene-expression profiling (Couillard-Despres et al., 2006; Brackoet al., 2012).
Constitutive and inducible reporter lines and viral systems to a large degree complement each other for the purposes of genetic, molecular, and physiologic analysis of adult neurogenesis. Table 1 illustrates the strength and weakness of the available and to be developed (shaded) methods for ontogenetic cell labeling.
(G. Enikolopov et al., 2015)
1.2 Inflammation in the nervous system
Now a day there is a focus towards to study the cross talk between neuronal cells and immune cells followed by acute and chromic inflamamtoy conditions. Mnay reports are available to study the inflammatory molecules by in vivo and in vitro studies of lymphocytes and monocytes that contain the subsets of inflammatory, mediators that will give protection to the brain. Cells involved in the neuroinflammation and neuroimmune activation are microglial cells, mast cells, macrophages and ependymal cells (Kettenmann et al., 2011).
In the healthy brain, trafficking of cellular and molecular components from the peripheral circulation is regulated by the blood–brain barrier (BBB). However, after brain injury, the tight junctions between endothelial cells of the BBB become permeable, allowing peripheral immune cells to infiltrate brain parenchyma (Vasilache AM et al., 2015). These cells, along with the proinflammatory cytokines they secrete, contribute to the inflammatory response that follows brain injury. Acute inflammation arises both from responses of resident immune cells, the microglia, as well as from infiltrating immune cells of the peripheral circulation. Inflammation of the mammalian brain, however, is not only a response mechanism that follows CNS injury, such as from traumatic brain injury, spinal cord injury, and stroke. Recent studies suggest that inflammation plays a crucial role in neurologic diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Parkinson's disease (Tobin MK et al., 2014).
1.2.1. Microglia
Microglia cells are resident macrophages of the brain (Lawson et al., 1990). Microglia is participated in the immune defense and homeostasis of the nervous
microglia participate in the process of wiring the forebrain circuits, monitoring the growth of dopaminergic axons in the forebrain laminar positioning of subsets of neocortical interneurons. Microglia transforms their morphology in physiological and pathological conditions. In physiological condition microglia acts as a resting state in the central nervous system. In this condition microglia looks like a small cell body with elaborated thin processes.
After an injury, the brain microglia change their morphology from ramified form into amoeboid. This event is known as microglial activation. In the amoeboid form, microglial cells and their processes, increase in the size and started to produce immune molecules. If insult to the brain persists, microglial cells transform further into phagocytes. Activated microglial cells function in quite heterogeneous way in the different parts of the brain. The exact mechanism of microglial activation is not yet fully understood. One of the possible mechanisms by withdrawing the molecules released during normal brain activity or else by the production of abnormal molecules (Davlos et al., 2005 ; Raivich et al., 2005).
1.2.1.1 Microglial migration
In the pathophysiological processes, microglial migration is necessary for immune defense and wound healing. Two types of movements occur in microglia. In the activated form of microglia or non-activated state microglial cells actively move their processes without translocation of the body. In the amoeboid form or active state entire microglial cell migrate through the brain tissue (Davlos et al., 2005).
Microglial cells are considered as important phagocytic cells of the brain helps in brain development, pathology and regeneration. During the CNS development, microglial phagocytosis is crucial in removing apoptotic cells and involved in synapse clearence during development and potentially pruning synapses in the
1.2.1.2 Histological studies of Microglia
Using advanced microscopical methods distinctive states of microglia in the physiological and pathological brains were revealed. In physiological conditions microglia have long processes and many branches and have a ramified morphology. Microglias are found in the various parts of the brain like hippocampus, subtantia nigra, basal ganglia and cerebellum. Gray matter contains more microglia than white matter.
In the pathological conditions, microglia loose thier processes and their nucleus is more enlarged in shape and they found more in the injury site. (Kaur et al., 2001; Zusso et al., 2012)
Microglia exhibit four different functional behaviors: (i) surveillance, (ii) neuroprotection, (iii) phagocytosis, and (iv) toxicity. Historically, most studies have focused on a detrimental role for microglia in the adult brain. The removal of microglial cells in the cental nervous system is usually described as destruction. Since it is difficult to distinguish between both destructive and phagocytic microglial cells, there are few evidences available to show that microglia are primary mediators of brain damage in vivo (Graeber 2010; Biber et al. 2014).
1.2.1.3 Microglial phagocytosis is essential for maintaining CNS homeostasis
Microglia does remarkable function of removing debris and dead neurons that may leads negative impact on brain function. There are enormous benefits of microglia-mediated removal of debris in brains of individuals with neurodegenerative diseases. Best example to show the beneficial effects microglia by understanding the pathogenesis of Rett syndrome (Derecki et al., 2012). Rett syndrome is characterized by Mecp2 mutations on the X chromosome (Amir et al., 1999). When the Mecp2 mutation is resolved in microglia, either by bone marrow transplantation (head irradiation required) or by targeted expression of wild-type gene Mecp2 in microglia of the mice with a Cre-LoxP system. The disease related symptoms are markedly
activity is pharmacologically inhibited in vivo (Derecki et al., 2012).
1.2.1.4 Microglia suppress destructive inflammation
It is now well accepted that inflammation all chronic neurological diseases. the mechanisms that the body has adopted to regulate or rectify the inflammation by producing inflammatory mediators such as IL- 4, IL-10, and TGF-b. These anti-inflammatory mediators suppress the function of pro anti-inflammatory cytokines
1.2.1.5 The M1 and M2 microgla phenotypes
Myeloid monocytes can be polarized into pro-inflammatory (classically activated, or M1) or anti-inflammatory (alternatively activated, or M2) phenotypes (Gordon, 2003). Recent reports suggest that M2-phenotype microglial cells are neuroprotective (Kigerl et al., 2009; The molecules produced by microglia do not often fall conveniently along the spectrum of the M1 versus M2 classification Moreover, the expression of a limited number of M2-related genes cannot reliably describe the entire function of microglia (Chen et al., 2012; Miron et al., 2013; Stirling et al., 2014). By production of pro inflammatory cytokines such as IL- 6, IL-12 and TNF-α, generating, ROS considered M1 phenotype as a neurotoxic. (Oliveira AFM et al., 2011) The present challenge we are facing today is to associate the molecules produced by microglia with their potential protective functions.
1.2.1.6 Microglial Involvement in Adult Neurogenesis
Neurogenesisis modulated by a series of physiological and pathological conditions, acting through a variety of cellular and molecular mechanisms (Marin-Burgin and Schinder et al., 2012). Microglia is historically implicated in regulating immune and inflammatory response to pathological conditions. In recent years, more active research on microglia recognized it is an essential component of adult neurogenic niche.
In this hippocampus, microglias are physically part of the niche and intermingle with all cell types, and phagocytose the excess of newborn cells that undergo apoptosis in physiological conditions. (A Sierra and M Tremblay et al., 2014)
The first evidence for phagocytic role of microglia comes from studies showing that mediation of inflammation in adult hippocampal neurogenesis considered as determintal.LPS induced inflammatory challenge shown to decrese the BrDU-labeled neurons and this reduction were reversed by administering anti-inflmmatory drug indomethacin (Ekdahl et al., 2003; Monje et al., 2003). LPS participate in the initiation of synaptic connectivity of the embryonic neurons at the early stages of their synaptogenesis (Chugh et al., 2013) and inhibits their reactivation during spatial orientation (Belarbi et al., 2012).
An in vitro study also supports the fact that LPS treated microglia medium, downregulates the neurogenesis by upregulation of pro inflammatory cytokines. (Monje et al., 2000). At the same time, IL-1β upreulates the proliferation of cultured human hippocampal newborn neural progenitors, but attenuates their differentiation into neurons (Zunszain et al., 2012).
1.2.1.7 In vivo imaging of microglia in neurodegenration
In the Alzheimer disease, increased number of microglia has been identified around the plaques. Mayer-Luehmann et al. in 2008 showed the formation of new plaques in semi quantitative way that microglia were rapidly recruited to the newly formed Aβ plaques. Bolmont et al., in 2008, showed the relationship between microglia and plaques in APP/PSI transgenic mice crossed with the IBA1/GFP microglial reporter mice. First the microglial processes tend to move towards the plaques and followed by pulling them along the axis of the processes towards the plaque deposits.
1.2.2 Inflammation in stroke
schemic stroke is the third leading cause of death in western industrialized countries and a major cause of long-lasting disability (Dirnagl et al., 1999, Lo et al., 2003). It kills about 16 000 Canadians every year and approximately 300 000 Canadians live with the effects of stroke. In all instances, stroke ultimately involves dysfunction and death of brain cells, and neurological deficits that reflect the location and size of the compromised brain area. At present, the clinical treatments remain poorly effective (Dirnagl et al., 1999, Lo et al., 2003). The only approved therapy is trombolysis
induced by intravenous administration of recombinant tissue plasminogenactivator
(tPA), however because of a short therapeutic time window only a small fraction of patients can benefit from it. According to a current view, treatment of stroke is suboptimal without combination of clot-lysing therapy with neuroprotection and/or pro-regeneration treatments (Grotta 2001, Gladstone 2002, Lo 2003). Experimentally and clinically, stroke is followed by acute and prolonged inflammatory response characterized by the activation of resident glial cells, production of inflammatory cytokines and leukocyte infiltration in the brain, events that may contribute to ischemic brain injury (Dirnagl et al, 1999; Lo et al, 2003, Kriz J 2006). However, whether inflammatory processes are deleterious or beneficial to recovery is presently a matter of debate and controversies. Brain damage following transient or permanent ischemia results from a series of pathophysiological events that evolve in time and space. The early events are characterized by the acute energy failure and peir-infarct depolarization. This is followed by excitotoxicity and oxidative stress (Dirnagl et al., 1999, Lo et al., 2003). Inflammation is part of a delayed tissue response to ischemic injury. The inflammatory response following cerebral ischemia has two components: the inflammation of the blood vessel wall and the inflammatory response of astrocytes and microglia, the resident brain cells. After the onset of blood vessel occlusion, the ischemic injury triggers inflammatory cascades in the parenchyma that further amplify tissue damage (Stoll et al.,1998). The whole inflammatory cascade is initiated few hours after initial stroke and may last for several weeks and/or months after initial injury (Lo et al., 2003, Lalancette et al., 2009). Many evidences suggest
that post-ischemic inflammatory response may markedly affect injury induced neurogenesis in the brain (Liu et al., 1998).
After induction of stroke many inflammatory cells, including neutrophils, macrophages, and T cells, are recruited to sites of ischemic injury. The mechanism by which these cells decrease the neurogenesis after ischemic injury, and the way in which they affect NSCs, remains hugely unclear. Post-ischemic inflammation is characterized by microglia activation followed by infiltration of circulating inflammatory cells (Hermann DM et al., 2014). Acutely, reactive oxygen species and the inflammatory mediators cause endothelial cell and leukocyte expression of adhesion molecules, promoting the adhesion and migration of circulating leukocytes, which leads to a rapid inflammatory state at the site of injury. Leukocytes release inflammatory cytokines that lead to tissue damage at the site of injury and the ischemic penumbra (Tobin MK et al., 2014) (Figure 8).
Figure 8 Inflamamtion leads to secondary brain inflammation and cell death after ischemic injury
barrier (BBB) dysfunction with loosening of cell-to cell tight junctions between endothelial cells and up regulation of endothelial cell expression of various cell adhesion molecules (Tobin MK et al.,).
Neuroinflammation after ischemic stroke is self limiting event at most times. However, unlike systemic inflammation that subsides the exhaustion of inflammatory mediators, resolution of inflammation in brain seems to be an active process in which inflammatory mediators are suppressed by regulatory mechanisms (Iadecola C et al., 2011). Still there is debate about weather post-ischemic inflammation is benfeical or detrimental to neurological disoreders.
Severity of stroke is asscoated with highles levels of IL-6 presence in the blood circulation has been identified. Increase of cererbral inferction followed by injection IL-1B cytokine ijection and interleukin-1 receptor antagonists reverese this action was identied in expreiments conducted in rat brain. (Kriz J et al., 2006).
Minocycline a known anti-iflammatory/anti-microbial drug helps in neuroprotection after cerebral ischemia.
Treatment for cerebral ischemia by anti-inflmmatory drugs helps in neuroprotection by blocking infllmmmatory molecule cycloixygenase2. Collectively these results suggest that overactivation of inflammation causes brain damage, and modulation of infllamtion by anti0inflmmatory drugs will be useful in the treatement of ischemia (Savitz J et al., 2012).
In the brain microglial cells are play a major role in immune system .. Earlier studies are shown that microglial cells induce neuronal death by producing cytokines, but recent studies proved that microglia may participate in the neural recovery after ischmic injury in the brain by releasing growth factors. (Lull ME et al., 2010).
successful brain repair. The deeper understanding of the process of how the inflammation increases the susceptibility towards premature neural cell death will help us in a better way to design therapies to promote brain repair (Kriz J et al., 2006).
1.2.3 Neuroglial inflammation in ALS
Involvement of glail cells in the central nervous system of various transgenic mouse models of ALS were already reported (Kawamata et al., 1992). Levels of cytokine from the cerbro spinal fluid of ALS patients revealed that CCL2, CCL4, CXCL8, CXCL10, IL-1, IL-7, IL-9, IL-12, IL-17, IFN- and TNF- were elevated. Increased level of cytokines is correlated with disease severity and neuroprotectiveness.
1.2.3.1 Microglia in ALS
Post-mortem tissues of ALS patient brains showed that prescence of activated microglial cells in the anteirion horn of the spinal cord. Activation of microglial cells in the motor cortex of thalamus also seen by neuroimaging studies. In presymptomatic satge G93A mSOD1 transgenic mice brain exhibits microglial activation followed by neuroonla death (Yamasaki et al., 2010; Kawamura et al., 2012).
1.2.4 Cytokines and Neuroplasticity
Cytokines are produced because of immune response by different type of cells in the body, including white blood cells. (Dinarello C A et al., 2000). Both Interferons(IFNs) and Tumor necrosis factors are known to induce immune cells like natural killer cells and macrophages (Fensterl V et al., 2009), these cytokines are responsibele for cell death (Sun M et al., 2007). In the central nervous system, these inflammatory molecules play a crucial role in removing neuronal cell debris, as well as exerting physiological and neuroprotective functions (Pan W et al., 2001).
cytokines exerts positive effects to neural progenitor cells and modulate neuronal proliferation and neurogenesis (Figure 9 & Table 3).
Figure 9 Cytokines in an adult brain and thier influence in neurogenesis
After the induction of inflammation microglia and the immune cells produce cytokines that activate signaling pathways, they modify the adult SVZ and SGZ progenitor cells (Gonzalez-Perez et al., 2012).
