Thank you for your interest in our work. We appreciate all the comments and the necessity for a healthy debate on what is a controversial topic with significant potential implications for the management of MS patients.

In addressing some of the comments, we believe it is necessary to clarify the initial goals set forth in designing our experiments. We are well aware of the limitations of animal studies, as most people in the scientific community. However, while they have their limitations, animal studies enable experiments to be done that would otherwise not be feasible or ethical in human investigations and clinical trials. In designing our studies, our aim was to create an animal model of CCSVI and try to establish a cause and effect relationship to support the purported link to demyelinating diseases. In simple words, can jugular stenosis with secondary altered cerebral hemodynamics result in demyelinating plaques? We had no particular preference for outcome and, if anything, a positive link would have been much more simple to interpret and publish than a negative link.

We clearly demonstrate that the JVL procedure resulted in jugular stenosis. Furthermore, we performed nuclear scintigraphy experiments to confirm that our JVL model resulted in altered cerebral hemodynamics, a hallmark of CCSVI observed in MS patients (Zamboni et al., 2009). Indeed, upon quantification of the radioactivity of the excised, [saline-perfused] brains, the JVL group exhibited cerebral hypoperfusion when compared to the sham group, which had undergone the same experiment in parallel.

An EAE group was not included in the nuclear scintigraphy experiment since this is an established murine model of MS based on immunization against myelin antigens. Although this would be an interesting experiment, we do not believe that its results would significantly alter our conclusions. Indeed, even if there were altered cerebral perfusion and/or jugular vein stenosis in the EAE group, given the absence of any evidence of demyelination in the JVL group, these results would only suggest extracranial jugular stenosis and cerebral perfusion changes are an epiphenomenon or secondary effect and not the primary cause of demyelination.

As for the arguments indicating the difference in mice and humans, we are fully aware of the limitations of animal experiments as discussed above. However, in order to try to create a model that is as representative as possible, we not only confirmed extracranial jugular stenosis in the mice but also showed alterations of cerebral hemodynamics, similar to that reported in CCSVI (Zamboni et al., 2009).

As a note of clarification, we felt it necessary to indicate that “Dawson’s fingers” are an imaging finding. These can be seen and may support the diagnosis of MS but in isolation are not specific for it. Pathologically, there is ample evidence for neuroinflammation in MS plaques in humans (Gray et al., 2008). To increase the sensitivity of our studies, we evaluated the mice with molecular imaging, flow cytometry, and histopathology, but could not find any imaging or histopathologic evidence of demyelination or neuroinflammation in JVL mice.

The issue of chronicity in our JVL model was one that we took seriously in our experimental design. It is for this reason that our study was carried out until 6 months post-jugular vein ligation for our more sensitive experiments assessing demyelination and neuroinflammation, with the earlier endpoint of 4 months for confirmation of our JVL model. The 6 month post-jugular vein ligation time point (with ligation at 2-3 months of age) was chosen based on the average life expectancy of female SJL mice (395 days) (Storer, 1960), as well as the typical age of onset of MS in humans (20-30 years old) and an average human life expectancy of 77.9 years. Thus, at the 6 month post-ligation time point, the mice were ~250 days old with ~180 days of ligation which is a significant length of time considering the female SJL mouse life expectancy. Furthermore, SJL mice exhibit a high gross tumor incidence with 90% of this particular strain developing reticulum cell sarcomas by 13 months of age (Fujinaga et al., 1970). Taking all these factors into account, we believe that our JVL model is sufficient in terms of chronicity while also avoiding extraneous pathologies exhibited in older SJL mice. Indeed, some MS patients have a substantial burden of plaques in their brains at the time of initial diagnosis and the development of demyelinating plaques can precede clinically diagnosable disease by many years. Therefore, we feel that the complete absence of any evidence of neuroinflammation in these mice is unlikely to be related to insufficient chronicity of JVL.

We would like to thank everybody for their interest in our work. Given the conflicting reports and potential confounding factors in clinical studies, in particular the placebo response, it is likely that the CCSVI hypothesis and treatment will have to undergo a blinded, placebo controlled trial to determine the validity of the hypothesis and efficacy of the proposed treatment regimens.

Wendy Atkinson
Reza Forghani
John Chen


References:

Zamboni P, Galeotti R, Menegatti E, Malagoni AM, Tacconi G, et al. (2009) Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis. J Neurol Neurosurg Psychiatry 80: 392–399.

Gray E, Thomas TL, Betmouni S, Scolding N, Love S. Elevated myeloperoxidase activity in white matter in multiple sclerosis. Neurosci Lett 2008a;442:195-8

Storer J. B. (1966) Longevity and gross pathology at death in 22 inbred strains of mice. J. Gerontol. 21, 404-409. \par

Fujinaga S., Poel W. E., Williams W. C., and Dmochowski L. (1970) Biological and morphological studies of SJL/J strain reticulum cell neoplasms induced and transmitted serially in low leukemia-strain mice. Cancer Res. 30, 729-742. \par