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Thread: Cell therapy using bone marrow stromal cells in *chronic* paraplegic rats

  1. #1

    Cell therapy using bone marrow stromal cells in *chronic* paraplegic rats

    Cell therapy using bone marrow stromal cells in chronic paraplegic rats: Systemic or local administration?

    JesĂşs Vaquero, Mercedes Zurita, Santiago Oya and MartĂ*n Santos

    Neuroscience Research Unit of the Mapfre-Medicine Foundation, Neurosurgical and Experimental Surgery Services, Puerta de Hierro Hospital, Autonomous University, San MartĂ*n de Porres, 4, 28035 Madrid, Spain

    Abstract
    Recent studies showed the therapeutic effect of bone marrow stromal cells (BMSC) after spinal cord injury (SCI). In the present study, we compared the effect of systemic and local administration of BMSC in adult Wistar rats suffering chronic paraplegia as consequence of severe SCI. Adult Wistar rats were subjected to a weight-drop impact causing complete paraplegia, and 3 months later, all the animals remained without signs of functional recovery. At this moment, 3 × 106 BMSC were injected intravenously (n: 20) or into traumatic spinal cord cavity (n: 20). Outcome was evaluated until sacrifice of the animals, 6 months later, using the Basso–Beattie–Bresnehan (BBB) score, the cold spray test, and measuring the thigh perimeter. After sacrifice, samples of spinal cord tissue were studied histologically. The results showed that intravenous administration of BMSC achieves some degree of functional recovery when compared to controls. Nevertheless, administration of BMSC into postraumatic spinal cord cavity promotes a clear and progressive functional recovery, significantly superior to the recovery obtained by means of the intravenous administration. This effect is associated to long-term presence of BMSC in the injured spinal cord tissue, with images suggesting neuronal differentiation and spinal cord reconstruction.

    Keywords: Bone marrow stromal cells; Stem cells; Spinal cord injury; Paraplegia
    Dr. Young, I do not have access to this journal. Would you mind getting a copy and summarizing the results for us?
    ...it's worse than we thought. it turns out the people at the white house are not secret muslims, they're nerds.

  2. #2
    Senior Member roshni's Avatar
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    Wish I knew a little more about the outcome measures - "cold spray test"?

  3. #3
    Steven, I will try to get a copy of the paper. Questions not answered in the abstract include:
    1. What kind of cells did they isolate from the bone marrow? Did they select cells for specific markers, i.e. CD34, CD44, etc.
    2. Were the cells from other animals or autografts. If the former, did they use cyclosporin.
    3. What strain of rat was used?
    4. Did they look for and find regeneration or myelination?
    5. What marker did they use to identified transplanted cells?

    Wise.

  4. #4
    Wise, thanks. I am curious as to what they meant by "significant".
    ...it's worse than we thought. it turns out the people at the white house are not secret muslims, they're nerds.

  5. #5
    Steven, I just read the paper. Here are some relevant details.
    1. Bone marrow stem cells. The authors obtained the cells from Wistar rats (12 weeks old) from bone marrow, cultured them in 20% fetal bovine serum for 3 days, removed nonadherent cells, and replaced medium every 3 days. When the cultures reached confluency, they tagged the cells with Hoechst 33342 (a nuclear label), rinsed them, incubated in trypsin for 5 minutes (to suspend them), and the injected them either intravenously or intraspinally. Note that they did not use cyclosporin or other immunosuppressants (this is strange because the bone marrow cells are very likely to have been rejected).
    2. Spinal cord injury. They used 50 female Wistar rats (different from the ones that donated the bone marrow), did a laminectomy at T6-8, injured the cord by dropped a 25 gram weight 20 cm (by the way, I think that this was a mistake... they probably meant 20 mm because if they had dropped a 25g rod 20 cm, it would sever the cord). At 3 months after injury, they transplanted 3 million through a tail vein or into the spinal cord injury site (incidentally, it is curious that they used the same dose of cells both for intravenous and intraspinal because 3 million cells for intravenous is too low).
    3. Outcome measures. For 3 months after the transplant, the authors obtained daily BBB scores and sprayed ethyl choloride (which cools or perhaps even freezes the skin) and observed the animals for local skin twitch, transient vocalization, and sustained vocalization (presumably because the rats can feel the pain). They used the Student's t-test (which may not be the appropriate statistical test for repeated measures) and the behavioral tests were done daily (which is a lot). For histology, they did frozen sections and H & E stain, immunohistology for neurofilament.
    4. Motor Recovery. Although the authors say that they did daily BBB scores, they only showed monthly scores. The results are puzzling. In the 3-month intraspinal transplanted animals, they recovered to BBB scores of 6 by 1 month and 8-9 by 3 months. After transplant, they recoverd to what appears to be about 12-13. This is reasonable. However, the intravenously treated rats showed no recovery before the transplants, achieving BBB scores only to about 1 at 3 months and remained at less than 2 even after the intravenous transplants. Why should there be such a big difference between the BBB scores of the animals before transplant at 3 months? Clearly, this must be an error.
    5. Sensory Recovery. Again, the authors show monthly cold spray tests. In the intraspinal bone marrow transplanted animals, the animals showed recovery up to 2.6 or higher by 3 months (2 meaning transient vocalization). After the intraspinal transplant, the scores increased to about 2.7 (3 meaning prolonged vocalization). In contrast, animals that received intravenous bone marrow cells had zero scores up to 5 months and had some recovery to a score about 0.7 at 6 months. Again, there must be some kind of error here. Why should there be such a big difference between the sensory scores of the animals before transplant at 3 months?
    6. Histology. The authors show one presumably representative H&E stained section from a control rat and one from an animal that was treated with intraspinal bone marrow. They suggest that there are tissue bundles that traverse the lesion site. In my opinion, the images are too poor to allow that condition. Many contused spinal cords do fill up with a loose matrix of tissue by this time (we reported this by the way over 700 contused rats in 1997) and many axons are present in the loose matrix. The authors claimed that there were hoechst stained (bisbenzimide) cells at 6 months but I don't believe these results because if the cells die, the dye will be taken up by other cells. That is why the Hoechst stain is not a good label of visualizing long-term transplanted cells.


    I am frankly very disappointed in this paper. It must have been reviewed by somebody who does not know the spinal cord injury field and did not pay attention to the data that was being presented. The description of the methods must be wrong and the data does not make sense. I have read it several times and think that the description of the injury method must be mistakened and the results are not credible. These errors should have been have been detected during the peer-review process. Any knowledgeable reviewer would have seen these problems and should not have approved this paper for publication.

    Wise.

  6. #6
    Wise, thanks for the reply.

    Is it too much to ask that journals publish accurate results and have competent reviewers?
    ...it's worse than we thought. it turns out the people at the white house are not secret muslims, they're nerds.

  7. #7
    Quote Originally Posted by Steven Edwards
    Wise, thanks for the reply.

    Is it too much to ask that journals publish accurate results and have competent reviewers?
    Steven,

    I want to correct my critique below. The authors of the paper had transplanted the rats at 3 months after injury and showed the results of the BBB and cold spray test monthly for 6 months after transplantation. So, the data of the first three months, which I had assumed was pre-transplantation, was not really pre-transplantation. I retract that objection although I would have liked to have seen what the pre-transplantation recovery of the animals were in the intraspinal and the venous infusion groups.

    I want to point out another puzzling part of the methods section. In the paper, the authors pointed out that they injected 50 microliters of cell suspension into the spinal cord. I think that this must be in error as well for the following reasons. First, 50 microliters is 50 cubic mm. That is a huge volume to be injecting into the thoracic spinal cord, which is a cylinder that is approximately 3.5 mm in diameter normally. I don't think that is it possible to inject that large an amount into the spinal cord. Second, one usually has 200,000 cells per µliter in a typical suspension. Perhaps they injected 15 microliters of cell suspension rather than 50 microliters.

    Concerning what to do about papers that one thinks have flaws, one has to write the journal and, if they consider the comments to be meritorious, they will publish the letter. That is the usual route by which one discusses papers that have been published. It is the same, I suppose, as when one sees an inaccurate article being published in the New York Times or other newspapers. Readers should comment if they there is an error. I need to think about this a bit more and then write something to the journal.

    Wise.
    Last edited by Wise Young; 01-23-2006 at 06:41 PM.

  8. #8
    Senior Member Buck_Nasty's Avatar
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    Now if they could only figure out how to turn us into rat's.

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