3. ANNEXES (Moo transcripts)

3-2 BioMoo Seminar

SEMINAR (5th NSJC, May 10, 1995. )

Gustavo turns the seminar recorder on.
Gustavo says, "Now we're in the air. ;)"
Norm nods to Lurker,Carlisle, and EJP
Carlisle disappears suddenly for parts unknown.
Carlisle materializes out of thin air.
Gustavo [to Norm]: ok, you can start whenever it suits you.

Norm shows slide #1.


                 ..oOQOo.   ..oOQOo.
                /+!+!+!+:. .:+!+!+!+\
               (+!+!+!+!+. .+!+!+!+!+)
               |!+!+!+!+:| |!+!+!+!+!|
               .\\+!+!+!+] [+!+!+!+//.
              (|+!\+!+!+!] [!+!+!+/!+|)
                \_+!+:_)(= =)(_:+!+_/
                        \| |/
                         | |


Norm says, "Hello all, Welcome to the journal club.""
TJ thinks Norm's brain is commissurotomized
Norm says, "I appreciate your coming today....I just wanted to see if anyone else show up.."
Norm Chukles at TJ's reading of my coronal MRI...Missing it's corpus Callosum!
Norm says, "Anyone who would like to speak during the Journal CLub (JC) can do so at any time, but you must first type: request"
Norm says, "Not a Huge turn out, but High quality I can tell. Ok. Here is the article for today:"

Norm shows slide #2.

          Neuroscience Journal Club #5
Article: Regeneration into the spinal cord of transfected dorsal
  root axons is promoted by ensheathing glia transplants

Authors: Almudena Ramon-Cueto and Manuel Nieto-Sampedro.
  Neural Plasticity Department, Instituto Cajal,
  Madrid, Spain.
Journal: Experimental Neurology, 127:232-244, 1994

BioMOO Presentation by: David McKalip, M.D.(Norm), Division of Neurosurgery, University of North Carolina at Chapel Hill.


Norm says, "BTW, anyone who would like to see the actual figures from the articel may do so with their WWW browser:"
Norm says, "Just use it to get to the BIoMOO page and click on my poster."
Norm says, "The general topic again is on ways to promote/study regeneration in the CNS."
Gustavo . o O ( shortcut: open http://bioinfo.weizmann.ac.il:8000/33/view/4454 with your web browser )
The housekeeper arrives to cart Taffy off to bed.
Norm says, "THese authors were experimenting with a glial population from the olfactory bulb:"

Norm shows slide #3.


Goal: To determine if the ensheathing glia (EG) of the olfactory bulb can promote regeneration of Dorsal Root Ganglion (DRG) central axons if the EG cells are transplanted into the spinal cord.


Norm Allows everyone to read the slide. "Any questions about the goal of this study?"
TJ thinks Norm should remind everyone that olfactory cells can regenerate, probably the only cells in the mammalian CNS that can
Norm says, "okay then. Here's how they studied this:"
Norm agrees woth TJ.
Norm says, "Excellent point.  "
Norm holds for a second to hear any other comments/querstions.
Norm hears none and proceeds.

Norm shows slide #4.

1. 13 Adult Rat T10 dorsal root was transected and reapproximated with suture. 

2. Cultures of Olfactory Bulb Ensheathing Glial (EG) cells were 
purified and grown using standard techniques.

3. EG Cultures were transplanted into dorsal root entry zone to depth of 0.2 mm (30,000 cells in 3 ul). 

4. Lesion areas examined for EG cell survival, and length/location of any axonal outgrowth.


Norm says, "This slide demonstrates the techniques used by Ramon-Cueto and Nieto-Sampedro for this study."
Norm says, "Basically, T10 roots were transected and reattaced..."
Norm says, "Ensheathing glial cultures were created and transplanted into the spinal cord in the region of the DREZ."
Norm says, "THen axon growth and Ensheathing glia (EG) survival was examined."
Norm says, "Here are the various experimental groups usd in the study."
TJ says, "I'm unclear why they used NGF receptors as the criteria for ensheathing glial cells.  They did not seem to mention it, but I assume that earlier work had shown olfactory glia have NGF receptors?"
Norm holds and reads.
Norm hmmms..
Norm says, "I am not sure and I don't see a reference. let me see."
Norm says, "no, no reference. Interesting poiinbt though because:"
Norm says, "It implies that they may produce NGF, much like Schwann cells. Don't schwann cells also have NGF receptors?"
TJ says, "Oh, there's one: #53 Eur J Neurosci 5:1172, 1994."
Norm looks.
John has disconnected.
TJ thinks Norm is right about Schwann cell NGF receptors.
Norm says, "I see. I should take a look at that. I'll have to look into the Schwann cell question."
Norm says, "Anyway, here's the experimental groups they used:"

Norm shows slide #5.


*Ensheathing Glial (EG) cultures:
     3 labelled with the nucleur fluorochrome: Hoesht 33342
     5 unlabbelled
*13 adult male Wistar rats
     5 control (lesions and not transplanted)
     8 experimtal
          3 transplanted with flourochrome labelled EG cells
               *These three examined with imunocytochemistry               (anti-Growth Associated Protein (GAP))
          5 transplanted with unlabbelled cells
               *These 5 examined with DiI labelling
                to determine axon extension


Norm says, "13 animals total with 5 controls (non-lesioned/non transplanted), 8 experimental.."
Norm says, "The EG cultuers were mainly unlabelled (n=), but 3 had a labelled nucleus."
Norm . o O ( unlabelled EG = 5. )
Norm says, "the 8 experimental animals (lesioned/transplanted) EG transplants: 5 unlabblelled and 3 labelled."
Norm says, "Three Weeks latere, the Spinal cord were examined."
Norm says, "Before we go into their results, I just wanted to review the Rexed's lamina of the Spinal cord gray matter:"
WatsonCrick (working on mcd) has arrived.

Norm shows slide #9.

--- /I \               /  \    DREZ = Dorsal root
    |II|               |  |           entry zone
    |III\             /   |   
    | IV \           /    |   I-V: touch, pain, temp. 
    |  V  \ ________/     |   VI: proprioception
     \  VI     X         /    VII: dorsal-spinocerebellar tract
     /       ----        \         Intermediate- autonomic
    /        |  |         \   X:   autonomic/pain
   /   VII   |  |          \  IX: medial-axial muscles
  |IX     VIII  |           |     lateral- limb muscles      
  |  IX    IX|  |           | VIII: Receives descending axons   
   \______VIII   \__________/        from brain.       

Norm lags
Norm Smiles at Wats. "Hey there."
Norm says, "As you can see, the dorsal horns are mainly sensory, the intermediate autnomic and the ventral motor."
Norm says, "Now, here is a summary of the ingrowth seen after transplantation:"

Norm shows slide #6.

        CONTROL (n)                     LESIONED (n) 
DiI:    Up to DREZ (3)                  Lamina I, II, III, IV, V (5)(ILDH)

GAP 43: Lamina I of ILDH (2)            Lamina I-V (3) (ILDH)
                                        Dorsal commisure and medial 
                                        lamina IV and V in CLDH
IL=Ipsilateral, CL=contralateral, DH = Doral horn, DREZ= Dorsal Root 
Entry Zone, "Lamina" refers to Rexed's spinal cord gray matter laminae.

Norm says, "Oops, forgot to mention how they studied the axon growth:"
The housekeeper arrives to cart John off to bed.
Norm says, "For those spinal cords receiving _unlabelled_ ensheathing glia (EG), DiI labelling was used."
Norm says, "For those receiving flourochrome labelled EG, anti CGRP and anti GAP43 was used."
Norm says, "As you can see, the axons _appear_ to grow accross the lesion/Drez and into the dorsal horn and some into the Contralateral Dorsal horn:"
Norm shows a schematic:

Norm shows slide #8.

    /I \               / *\    DREZ = Dorsal root
    |II|               | *|           entry zone
    |III\             /  *|
    | IV*\           /**  |   
    |  V *\ ________/**   |   * = site of axon regeneration
     \  VI**   X  **     /
     /       ----        \  
    /        |  |         \
   /   VII   |  |          \  
  |IX     VIII  |           | 
  |  IX    IX|  |           |    
   \______VIII   \__________/     


Carlisle says, "Contralateral is a *long* way to go!"
Norm says, "The extent of axon growth is indicated by the asterisks.  THe lesion was on the right side (as you see it)"
Norm says, "Yes it is...if it is real!"
Carlisle says, "exactly"
Norm says, "If anyone wants to see a slide again, just say so...."
Gustavo sees a very long axon under the schematic. ;)
TJ thinks Norm is doing a groovy job with the ASCII art
Norm chuckles at the extracorporeal regeneration....
Norm says, "Took me about 3o minutes...I just did it on Word perfect and pasted it here..used the tinyfugue client and did an "/quote "'filename"."
Norm says, "anyway...."
Norm says, "The EG cells seemd to be related to the axons as you see here:"

Norm shows slide #7.


Hoechst 33342 labelled glia cells found in:
        Laminae I, II, III, IV 
        medial lamina V
        Dorsal Commisure

Not in:
        Ventral Horns
Appeared to be associated with:
        GAP-43 and CGRP + axons 


Norm says, "So,it appears that the EG have lead to axonal regerneation, however, the following questions come to mind:"

Norm shows slide #10.

                        QUESTIONS FOR DISCUSSION

1. Is the GAP and CGRP positivity related solely to transplanted axon growth?

2. Is putative axon growth due to ensheathing glia?

3. What molecular factors are responsible for putative axon growth?


TJ says, "OK, the $64,000 question: are these connections *functional*?  They never say, and it seems pretty important to me."
Pas materializes out of thin air.
Norm says, "They did not appear to test for function..."
Norm says, "That is always a drawback to many regeneration studies..."
Norm waves to Pas
Lurker says, "My question too.  Is lamina IV an appropriate target for the axons and which axons?"
Pas waves
TJ says, "Why T10, do you think?  If they had done lumbar cord, they could have tested for reflexes more easily, and I would be more convinced if they had some data on recovery of normal reflexes."
Norm says, "These axons alledgedly started from the dorsal root, croseed the DREZ and the lesion (gliotic tissue-not shown) and then entered the grey matter."
Norm says, "Lamina IV usually receives axons from:"
Norm says, "Dorsal root affernts"
TJ says, "However, the fact that the axons crossed a glial scar is significant...axons hardly ever do that in patients, do they Norm?"
Norm says, "And initiates spinothalamic tract."
Norm says, "No, not hardly ever..."
Norm says, "Unfortunaltey, they did not put pictures of that in their article."
Norm says, "I agree with you TJ on the reflex model..would have been very convincing then."
Norm says, "However, you think they still could have tested for pain senstaion:would have been hard though with a thoracic dxermatome."
TJ nods in agreement with *everything* Norm says, as usual
Norm says, "One other question:"
Norm says, "I think CGRP activity can be pretty non-specifically increased following a Spinal cord lesion:"
Lurker says, "I don't recall what all goes into the thoracic segments. Shouldn't the intercostal somatic sensory fibers be able to affect respiration?"
Norm says, "That may not have been a good way to check for ingrowth of axons."
Norm says, "However, GAP 43 is:"
Norm says, "The question then becomes, are these axons truly coming from the dorsal root or from local, intact tissue."
Carlisle says, "evoked potentials might have been a good way to test for functionality"
Norm says, "Yes, they may have been., but again, may have been difficult to localize it to one dermatome..."
Norm says, "You could do an open incision and put the electrode righyt on the nerve root then."
Carlisle says, "couldn't they have picked a better dermatome?"
Carlisle says, "yeah, norm!"
Norm says, "Maybe, I have not worked much with rat spinal cord, so I don't know if anatomy limits things."
Lurker says, "but if you want to do that then why not use the lumbar segments?  There the motor neuron pools are pretty well defined.  I don't understand why the thoracic segments were used."
Norm says, "I should have asked these folks to come to the meeting: We have the technology."
Carlisle says, "Yeah, the lumbar segs were the way to go."
Lurker says, "I don't even know if the thoracic segments have DRPs."
Norm says, "For the sake of argument, let's assume regeneration did occur, the question then becomes: why:"
Norm . o O ( DRP's? )
Norm says, "Would the presence or abscence of DRP's ahve affected things?"
TJ was just reading a paper from McGill that claimed myelin-associated glycoprotein (MAG) is responsible for inhibition of neurite outgrowth.  Anyone know if olfactory
Carlisle says, "presumably the glial cells are infiltrating the cut surfaces and releasing growth factors, right?"
Norm says, "I suppos that's one explanation."
Lurker says, "they would indicate functional connections and intact relexes without the necessity of looking specificly at motor responses."
Carlisle says, "and acting as a physical bridge, too."
Norm says, "If it is real though, I am biased to say it is due to ECM factors or cell surface antigens."
Norm says, "They point out that EG's produce laminin"
TJ finds himself agreeing with Norm again about the ECM/cell surface idea
Norm says, "and NCAM,L1,and fibronectin."
Carlisle says, "well then you ought to be able to just inject ecm molecules, right?"
Norm says, "I think the anatomical benefit of an *ENSHEATHING* glial cell may play a role too."
Norm [to Carlisle]: they are trying that here in our labs:not very successful
TJ thinks exogenous ECM may be degraded, it would be good to have cells making it.
Carlisle [to say]: Oh, yeah...
Norm [to TJ]: I agree completely.
Norm says, "That's why I want to introduce such molecules with viral vecotrs...."
TJ says, "This might be the connection between this paper and Rusty Gage's work we covered earlier, with the fibroblasts...they would both secrete ECM, right?"
Norm Smiles.
Norm says, "Well, Gages' fibroblasts excreted NGF..."
Norm says, "But, when I run these things I always let my bias of ECM's role be pretty transparent."
TJ in a mocking tone, says, "but that's not all they secreted, is it? :-)"
Carlisle jeez, we'd better get to the library and read Gage's paper.....  ;)
Norm says, "The authors do postulate a role for NGF here though...."
Norm [to Carlisle]: That is NSJC #1 I believe.
TJ is sorry, he thought Carlisle was *at* NSJC #1
Norm [to Carlisle]: you can get the transcript of that journbal club here!
Norm says, "The authors point to some circumstatnial evidence indicating EG cells produce NGF."
Norm says, "As far as an *ensheathing role*"
Carlisle says, "Oh, yeah, well, Alzheimer's is a difficult thing to deal with....  ;^)"
Norm says, "The authors speculate that enveloping of the regenerating axons was important."
Carlisle says, "Anyway, it's not like there aren't a zillion *other* growth factors that we could be considering"
Norm nods in agreement.
TJ says, "What about inhibition of axon outgrowth?  Don't some people think myelin-associated glycoprotein (MAG) inhibits axon outgrowth?  I think ensheathing cells of the olfactory bulb are more like Muller glia of the retina, just wrapping around axons without "
Norm says, "The author's point to the locations of the EG cells:"

Norm shows slide #7.


Hoechst 33342 labelled glia cells found in:
        Laminae I, II, III, IV 
        medial lamina V
        Dorsal Commisure

Not in:
        Ventral Horns
Appeared to be associated with:
        GAP-43 and CGRP + axons 


TJ says, "myelinating them, right?"
Norm says, "I guess a psuedo-myelination yes:"
Norm says, "They imply it will promote axon guidance: they also examine wheather these cells migrated after injection."
Norm says, "The points raised were: did these cells migrate before regeneration and the axons went where they were, or:"
Norm says, "Did they follow the axons  along: migrating with growing axons."
TJ . o O (hmm...)
Norm says, "back to the ensheathing point: Did ensheathing "insulate" then from local inhibitory factors?"
Carlisle . o O (cool idea)
Norm says, "Well, I still think it points to the role that the environment plays ion CNS regeneration (or lack there of)."
Irit goes home.
Norm says, ""TJ, I know you work in this field.  DO any of you other folks do reseach on th"
Norm . o O ( Research on this topic? )
TJ . o O ( think hmm^2 )
Norm Watches TJ's mind work.
TJ says, "No, we are interested in *normal* development, but have not looked at regeneration.  Yet. :-)"
Norm nods.
Carlisle says, "We (Steve, Melinda and I) gotta split...cool paper!  Sorry we have to leave.  Catch ya on the flip."
Norm says, "I think all of the molecular principles of the developing nervous system can be applied to overcoming the lack of regerneation in the CNS."
Norm waves. "Thanks for coming.
TJ waves goodbye to Carlisle, Steve and Melinda
Carlisle has disconnected.
TJ . o O ( I hope so )
Norm nods.
Norm says, "Well, any comments Lurker?"
Lurker says, "regeneration isn't my field. "
Norm says, "Ok, Well, It might be interesting to exmaine these cells in culture."
Lurker says, "what little I know I learned while at Calgary.  Stem cells and all that."
Norm says, "Using various antisense genes to ECM or other cell surface molecules."
TJ . o O ( Norm is getting ready to ask Lurker to do the next NSJC, I bet :-) )
Gustavo chuckles.
Norm [to Lurker]: OK, no biggy. I just wanted to giver everyone a chance since it looks like we are winding down.
Norm is pretty transparent.
WatsonCrick nods enthusiastically
Norm chuckles.
Norm [to WatsonCrick]: Were you enthusiastically nodding to do the next NSJC??????
WatsonCrick says, "of course :)"
Norm says, "Polly, do you think any of this can be applied to Cetacean studies?"
Norm Does a double take at Wats. 
Pas says, "certainly, I can't be specific since I haven't read this article yet :)"
Norm says, "Well, we shall see what develops. Unless there is more discussion, I supposw we can call it wquits?"
Norm nods to Pas with a smile.
TJ nods.  Good Journal Club, Norm!!!
Gustavo . o O ( as usual! )
Norm says, "thanks TJ. "Thankls' all for coming....Hope we can take something back to our labs from this.!"
Lurker says, "ciao!"
Lurker has disconnected.
TJ says, "Bye."
Gustavo turns the seminar recorder off.

VMDL/MOO Report - 17 FEB 1996

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