Repair of CNS injuries by transplantation of OECs

Question 1

what is so special about olfactory system?

Answer 1

it is the only area where neurons are continuously formed thoughout adult life. The newly formed axons are accompanied by special type of glia and grow into the CNS

Question 2

what type of glia accompany the forming axons?

Answer 2

olfactory ensheathing cells

Question 3

what was the general thought behind the OEC experiments?

Answer 3

• can you transplant OECs into regenerating axons to regenerate and restore function

Question 4

what 4 injury models can be used for testing OECs?

Answer 4

• corticalspinal tract lesions
• spinal cord hemi-section
• spinal roots lesion
• optic nerve transections and glaucoma model

Question 5

why are the cortical spinal tract region used as a model?

Answer 5

• there is an antibody that you can use so that you can easily measure the injury to the tract and how it regenerates

Question 6

how did they functionally check the outcome of CST injury?

Answer 6

• they lesion the rat and then put food on the platform so that a paw can reach through and take the food through the gap - - normally the rat will reach through with both hands
• they remove one side of the cortical spinal tract (it is present on both sides) so that they can compare to the control on the other side of the spine
• you see that the the non lesioned paw side is only used
• this shows that after this lesion the rat cant reach through and take the food with that paw

Question 7

what are the two sources of OECs?

Answer 7

olfactory bulb and olfactory mucosa

Question 8

what is the marker of OECs?

Answer 8

P75

Question 9

what mixture of cells is the best in terms of OECs?

Answer 9

culture for 2 weeks with a mixture ofFurthermore, it has been reported that a 50:50 ratio of OECs with olfactory nerve fibroblast-like cells, rather than pure OECs produces optimal transplant-mediated repair- but some people find that purification is best

Question 10

before you transplant, what is important n terms of labelling?

Answer 10

you transact with GFP marker so that you can recognise them in the transplant

Question 11

how long did it take after transplantation did it take for the axons to regrow ?in the corticosinal tract experiments?

Answer 11

around 3 weeks before was almost back to normal

Question 12

after they had transplanted and found improved function, how can you prove that it is due to improved on regeneration?

Answer 12

you use mmunostaining to label the CST fibres. you see that in the control thy stop at the edge of the lesion but when you transept yo see that the axons grow through the transplant and back to the original tract ad form the terminal field and they are being myelinated by the OECs and oligodendrocytes

Question 13

how are OECs similar to SC?

Answer 13

they myelinate axons

Question 14

what is a hemisection?

Answer 14

cut had of the entire spinal cord

Question 15

how can you ensure that the cells dont move away from the area of cut?

Answer 15

you can put them in a gel and be retained in that region

Question 16

how did they measure the effects of OEC transplant onto a hemisection injury?

Answer 16

they added OECs in a gel and then measured the spriatory rythym from the phrenic nerve - they found that the unrelated there was no activity bt in the treated the rhythm was restored and spontaneous

Question 17

what is the problem of wound son the DRG?

Answer 17

a glial scar formed by astrocytes

Question 18

how did they investigate the role of OECs n DRG damage? including how they tested for functional repair?

Answer 18

they cut and then transplant he OEC and label the axons- in the control you never see any axons crossing the border between the doral horn and dorsal cord but in the transplant and you can see that the axons cross the border.
- they looked at the rat moving up the poles of a cage.
- if you cut three roots C6-T1 you see a deficit in climbing and mistake made when climbing the bars.
you see with the transplant into the root that you get a increase in mean grasp score increased from 0-4 and the wild type is 7
they record from the cord dorm and cuneate and fun that the response to stimulus comes back (?)
- when you label with GFP you see that when you put cells in the dorsal root then they are more likely to bridge the gap to the spinal cord which doesn’t happen in controls

Question 19

how do they create a model for retina ganglion cell transection ?

Answer 19

• they add a cut to the optic nerve

Question 20

what is normally seen when the optic nerve is tansected?

Answer 20

axons are unable to regenerate across the cut and they actually retract 8 months after cut in the rat- but here are some remaining in the cut area- very few axons

Question 21

what happens when you put OECs into he region of optic nerve cut?

Answer 21

you see the axons crossing the cut region of injury and they are brought back to injury tract originally there

Question 22

what stimulated them to look at the role of OECs in repair after glucoma ?

Answer 22

• they saw that it could promote the repair of OECs

Question 23

where did they inject the OECs for the glucoma model study? what idd they see?

Answer 23

into the visual disc- they migrate into the retina na down into the optical nerve region. they also ensheath the RGCs and allow them to regenerate

Question 24

how did they mimic the intraocclar pressure caused in glaucoma? what did they find?

Answer 24

the injected magnetic microphones into the anterior chamber and recorder the IOP using a tonometer- found the beads increased the pressure . The OECs could not prevent the pressure but the found that the number of axons were increased in the OECs transplanted into the optic disc can reduce the loss of RGC axons caused by raised IOPe presence of IOP compared to without them this is not a clinical model because you inject the OEC before beads

Question 25

have OECs been used in humans?

Answer 25

• you can culture human bulb OECs
• they implanted this into the site of injury in the spinal cord
• they found that after 3.5 years and a half he could cycle around on a bike!

Question 26

name two highly occurring cause of non healing skin wounds

Answer 26

atherosclerosis and pressure ulcers

Question 27

why is looking at model organisms not that helping for wound heeling

Answer 27

• mode organism show a lot of variety in their abilities to repair wounds

Question 28

what is the first response to wounds?

Answer 28

• the clotting cascade forms a fibrin clot. Platelets trapped in the clot also release growth factors and cheekiness into the local wound environment

Question 29

in terms of inflammation, what is thought to be part of the cause of chronic wounds?

Answer 29

• they may be stuck in the pro-inflammation phase which become chronically activated: there is a continual competition between inflammatory and anti-inflammatory signals leading to a misbalanced environment for proper wound healing to occur.

Question 30

in terms of cells, which have bene implicated in chronic inflammation in chronic wounds?

Answer 30

• proinflammatory infiltrates composed largely of neurotrophils and macrophages contribute to delayed healing in chronic ulcers

Question 31

the deregulation of which prinflammatory cytokines have bene implicated in chronic wounds and were are these thought to come from? what do they do?

Answer 31

• IL-1beta and TNF-alpha, from macrophages and neutrophils they prolong the inflammatory response
• they are increased in chronic wounds and lead to elevated metalloporeinases hat degrade local ECm and prevent migration

Question 32

what exogenous factor promotes increased presence of pro inflammatory cells?

Answer 32

• an increased bacterial load

Question 33

how are proteases implicated in wound healing?

Answer 33

Unlike the acute wound-healing process, where tissue proteases are normally under tight regulation, it appears that in a chronic wound, disruption of the produc- tion and activation of proteases plays a role in wound pathogenesis. The proteolytic unbalance may be a consequence of the aforementioned faulty regulation of inflammation and/or microbial contamination. It has been shown that matrix metalloproteinases (MMPs), such as collagenase and gelatinases A and B, are elevated in chronic wound fluids when compared to acute wound fluid

Question 34

how are stem cells implicated in chronic wounds?

Answer 34

Recruitment of bone marrow and endothelial progenitors to the site of injury is coordinated by specific chemokines, which have been shown to be depleted in conditions that contribute to compromised healing response, such as aging and diabetes. Furthermore, frequent cycling of epidermal stem cells in patients with chronic wounds can lead to depletion of local stem cell populations. Compromised function of local and systemic stem cells and progenitors may play a considerable role in pathology of chronic wounds.

Question 35

how is angiogenesis implicated in chronic wounds?

Answer 35

nadequate local angiogenesis is considered a very likely contrib- utor to the impaired healing of DFUs (3). Proteins with antiangiogenic properties, such as myeloperoxidase, exhibit higher expression levels in chronic wounds of diabetic patients as compared to acute wounds, whereas angiogenic stimulators, such as extracellular superoxide dis- mutase, are generally decreased

Question 36

how are senescent cells implicated in chronic wounds?

Answer 36

One hypothesis is that cells, fibroblasts in particular, become prematurely senescent within a chronic wound setting. Senes- cence caused by oxidative stress might also drive uncontrolled fibro- blast proliferation and keloid formation (56). Senescent fibroblasts and keratinocytes secrete MMPs 2, 3, and 9, and might therefore exert an antifibrotic effect (57). Moreover, senescent keratinocytes have been documented to secrete the antiangiogenic factor maspin (58), which may also be detrimental to repair. They are also pro inflammatory

Question 37

what growth factors can be added to wounds and have been shown to promote wound healing?

Answer 37

FGF-2, KGF-1- not VEGF though

Question 38

what are 2 arguments against using growth factor application?

Answer 38

Given the complexity of the multicellular tissue repair response (Fig. 2), it is not surprising that therapeutic delivery of a single factor and/or cellular component often only achieves limited efficacy in stimulating healing of chronic wounds. Furthermore, recent studies indicate that cells within the chronic wound may lack other aspects of the molecular milieu that enable an appropriate response to growth factor stimuli, such as down-regulation of epidermal growth factor receptor (EGFR) and TGFb receptors as well as SMADs

Question 39

what is the most overt sign of chronic wounds?

Answer 39

failure of wound reepithelialisation

Question 40

what are the first genes to be upregulated at the wound edge epithelium? what will be the role of these genes?

Answer 40

• fos, ap-1, jun
• cell proliferation, epidermal migration of a leading tongue of keratincoytes at the interface between the scab and the healthy wound granulation tissue

Question 41

what needs to happens for the keratoncytes to migrate during wound healing

Answer 41

• some interns are switched off and others are turned on such as Beta1 integrin

Question 42

when repeithelialisation occurs, where do these stem cells me from it is thought?

Answer 42

he new keratncoyets are thought to come from the stem cell dense budge of hair follicles. ad from the non bulge region follicular cells.

Question 43

where do the wound fibroblast population come from, what showed this?

Answer 43

• fluor MSCs after intravenous injection were reported to contribute tot the wound fibroblast population - these are from the bone marrow normally

Question 44

what studies link inflammation to scarring?

Answer 44

Studies of repair in embryonic and fetal mice and also human patients undergoing fetal surgery have indicated that, before the onset of a wound inflammatory response, immature tissues are capable of scarless healing (99, 100). Inflammation might therefore be a cause of wound fibrosis. Indeed, mice lacking the ETS family transcription factor PU.1 (and thus not able to generate any leukocytic lineages) are able to heal wounds effectively as neonates—and do so without subsequent scarring, unlike their wild-type littermates

Question 45

what are the initial sources of inflammation?

Answer 45

Some of the earliest signals include factors released by degranulating platelets (23) and by damage- and pathogen-associated molecular patterns (DAMPs and PAMPs, re- spectively) where cells are damaged and microbes gain access
- s a wound gapes open and then begins to contract, focal adhesion kinase/extracellular signal–regulated kinase (FAK-ERK) leads to activa- tion of chemokine ligand 2 release by wound fibroblasts, which, in turn, draws in a larger inflammatory response

Question 46

why are flies good for regeneration studies? whats bad?

Answer 46

• good for tissue labelled- membrane labelling - translucency
• they only have a single immune cell lineage

Question 47

how have stem cells been used in wound repair? (2)

Answer 47

• Clinical studies have shown that bone marrow– and adipose tissue– derived MSCs can augment the repair process when applied locally to chronic skin wounds
• Recent developments in reprogramming skin and other differen- tiated cells into induced pluripotent stem cells (iPSC) provide a new cell source that can potentially be used for therapy. It has been shown that human skin equivalents can be created entirely from fibroblasts and keratinocytes generated from patients’ iPSCs, and that healthy cells can be generated from reprogrammed cells from patients with RDEB

Question 48

what are the 5 steps of wounding?

Answer 48

haemostasis, inflammation, proliferation and migration, followed by scar tissue remodelling

Question 49

how long after injury is cx43 upregulated? what is this correlated with?

Answer 49

• 6 hours

- keratinocytes crawling adopting a migratory phenotype as they crawl across the wound bed to close the epidermal breach

Question 50

what is upregulated in the leading edge keratinocytes during migration?

Answer 50

cx30 and 26

Question 51

what is the process by which cx43 is down regulated thought to be?

Answer 51

A role for protein kinase C has also been proposed in phosphorylating Cx43 serine 368 at the wound edge in order to decrease its communication,

Question 52

why is loss of cx43 from the migrating cells thought to be deseriable? (2)

Answer 52

• Cx43 can bind to an array of other junctional and cytoskeletal proteins, either directly via a PDZ domain or indirectly via other proteins such as membranous Cadherins, Zonu- lar Occludin-1 (ZO-1), α- and β-catenin, as well as cytoskeletal mi- crotubules and actin. Such interactions may affect both cell adhesion and cytoskeletal dynamics and therefore cell migration and wound healing.
  However, some of the interactions are with tran- scription factors such as β-catenin, and binding to it may keep β- catenin at the cell membrane, preventing it from influencing gene ex- pression. Indeed, it has been proposed that Cx43 forms the centre of a protein complex or “nexus” acting as a master gene that can influence expression of other over 300 other genes

Question 53

where is cx43 upregulated, why is this thought to be (mediated)?

Answer 53

within the first few hours it is upregulatedin the endothelial cells of blood vessels as they become inflamed- The upre- gulation of Cx43 is likely to be brought about by the proinflammatory signals being released at the wound site and this correlates with the blood vessels becoming leaky to leukocytes, proteins and fluids which in turn cause the dermis to swell.

Question 54

t are the steps of inflammation?

Answer 54

• mast cells and degranulating platelets form a fibrin clot and release PDGF which attracts neutrophils which express more inflammatory signal which in turn recruits monocytes that transform into macrophages

Question 55

what did people think about he inflammation response initially and what do they think now?

Answer 55

1. he production of a strong inflammatory response is thought to provide the signals to stimulate wound repair by transforming wound edge fibroblasts and keratinocytes into a migratory phenotype that will crawl forward to close the wound and fill the wound bed with granulation tissue
2. This suggests that far from being es- sential for triggering the onset of wound healing the inflammatory re- sponse, it may in fact be contributing to fibrosis and scar formation [57]. Indeed large numbers of neutrophils at a wound site may do more than just kill bacteria; the release of free radicals designed to kill bacteria may also kill healthy cells in the intact surrounding tis- sues

Question 56

how does application of nexxigon affect neutrophils?

Answer 56

neutrophils both in and around the wound sites on days 1 and 2 after injury are reduced following application

Question 57

draw a diagram to summarise the affects of antisense cx43

Answer 57

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Question 58

what evidence is there that the neutrophils and oocytes may not be needed for repair?

Answer 58

In the absence of both cell types, the repair of small wounds can still occur, and the scarring response is even less30.