Multiple Sclerosis, The Blood Brain Barrier, and New Treatment

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Multiple Sclerosis, The Blood Brain Barrier, and New Treatment

by R. Stout

Abstract: Survey of Journal articles shows preponderance of evidence that Multiple Sclerosis attacks occur during breakdowns of the blood-brain barrier. Other articles show that flavonoids including those found in blueberries and grape seeds amongst others can inhibit blood-brain barrier breakdown in rats under conditions which normally lead to such breakdown. The same flavonoids can also be effective in reducing inflammation, which might thus have value in treating the symptoms of an exacerbation. Suggested experiments are proposed to evaluate whether flavonoids may offer effective treatment for multiple sclerosis.

This article is highly technical, being intended for those with a background in biology or medicine. For a simpler version written for the lay person: Enter

Posted on the internet at http://spider.lloyd.com/~tstout/articles/multiple-sclerosis.html on June 14, 1996. This is a rewrite of earlier versions posted June 2, 1996 and May 23, 1996 .

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For a long time multiple sclerosis has been a puzzling disease. Recent experimental studies have clarified many of the problems associated with the disease.

Historically, it has been known that MS is caused by certain white blood cells attacking the myelin surrounding the nerve cells of a person's own central nervous system. This was looked at as a defect in the immune system and much work was concentrated in trying to explain MS simply as an autoimmune disease. However, the disease was much too erratic to fit the autoimmune models with any satisfaction.

Overlooked during these studies was a more fundamental question, "How did the white blood cells get to the myelin in order to attack it to begin with?" Under normal conditions the blood-brain barrier (BBB) provides an effective separation between the blood cells and the myelin, such that it would be irrelevant whether or not some of the white cells were programmed incorrectly. Hence, the question presents itself as to whether or not there is evidence of BBB malfunction during MS attacks.

Indeed, that is the case. On June 1, 1996 I did a computerized journal search at the University of California at Health Sciences Library with their biosis search engine. The search revealed 214 journal articles that responded to keyword searches for both Multiple Sclerosis and Blood-Brain Barrier.

An article published in March of 1996 demonstrates this new direction in which MS research is heading:

"Serial brain MRI at monthly intervals has provided valuable insights into the natural history of multiple sclerosis, and is now often used to monitor the efficacy of experimental treatments. Scanning at this interval often shows asymptomatic new lesions in relapsing-remitting or secondary progressive multiple sclerosis, on average five to 10 times more often than clinical relapse. About 80% of new lesions... show gadolinium enhancement..., indicating a breach of the blood-brain barrier.

"On weekly scanning, every new lesion on long TR images showed an initial phase of gadolinium enhancement.... Although we have only studied three patients, the consistency of this finding suggests that breakdown of the blood-brain barrier is an invariable and perhaps obligatory event in the development of new lesions in relapsing-remitting or secondary progressive multiple sclerosis." (Ref 1)

The body of the article discusses how that these three patients had 38 new lesions appear on MRI scans during the course of the study, and how that EVERY SINGLE new lesion was associated with a breakdown in the blood-brain barrier at the early stage. Thirty-eight instances without an exception is the consistency of which they spoke.

Although many other articles could be quoted tying together MS attacks and BBB breakdown, the above article expresses the matter quite clearly and for our purposes we will assume that further research will serve only to confirm and refine these findings and conclusions.

The true value of a good theory for the cause of a disease such as multiple sclerosis is the possibility of an effective treatment proceeding from the theory. In the light of the above discussion, a simple potential treatment presents itself: strengthen the BBB to the extent that no further breakdowns occur.

There are three related chemicals which have been found effective in strengthening the blood-brain barrier in animals. These are the anthocyanosides, proanthocyanidins, and procyanidolic oligomers (PCOs). All three of these are variants of a common class of chemicals called "flavonoids."

Anthocyanosides are the chemicals which give blueberries, cherries, and blackberries their color. Proanthocyanidins and their oligomers (PCOs) are found in purple grape skins and grape seeds and the bark and needles of certain pine trees. At the University of California at Health Sciences Library, there were two articles discussing the effect of anthocyanosides on the blood-brain barrier:

In one set of experiments the blood brain barrier was "opened" in test rats by placing various chemicals in the blood stream, such as proteases or DMSO. Then anthocyanosides were injected into the blood stream of some but not all of the rats. Some of the comments of the authors are "With all the four permeability increasing agents (proteases and DMSO) used in these experiments the O.D. of the dye extracted from the brains was lower in the anthocyanoside-treated groups than in the control groups. The permeability increasing effect of DMSO was nearly completely abolished. ... These results indicate that the anthocyanoside treatment diminished the permeability increase of BBB induced by proteases or DMSO." ... "As shown above, treatment with anthocyanosides diminished the permeability of the BBB to trypan blue after intraventricular injection of a high dose of collagenase." ... "Animals treated with the anthocyanosides recovered their normal BBB permeability in less than 24 hours in contrast to untreated controls who recovered only after about 72 hours." ... "Our results indicate clearly that anthocyanosides are able to act on the permeability of brain capillaries. The mechanism of this action deserves further attention." (Ref 2)

In an another set of experiments, rats were induced with experimental hypertension using an established procedure. One of the by-products of the procedure was an increase in the permeability of the BBB. During the experiments, some of the rats were treated with anthocyanosides. The experimenters' conclusions were: "Anthocyanoside treatment decreased the permeability of the (blood) vessel walls to the tracer in all our experiments... In experimental hypertension, the greatest reduction of permeability increase was observed in the cerebral vessels where the permeability was completely normalized by the drug." (Ref 3)

Discussion

In both of these experiments anthocyanosides were able to strengthen the BBB in rats. The question becomes whether or not such strengthening would be effective for human patients suffering MS lesions. The target goal for the MS patient would be to keep the BBB strong enough such that no future lesions would occur. If this can be realized, the goal of much MS research may finally be reached. It should be noted that these concepts are "leading edge"; it was only three months ago that the above journal article by Lei et al was printed (ref 1), in which evidence was given that in all 38 new lesions the researchers observed they also found evidence of BBB deficiency. The reason for this deficiency is unknown. Just because anthocyanoside treatment is effective in rats for the various mechanisms used to weaken the integrity of the BBB, this does not mean that it would necessarily be effective for MS. However, there were five different mechanisms used to degrade the BBB in the two articles we quoted and anthocyanosides were effective in strengthening the BBB in all five. So, because there were a number of different mechanisms used to degrade the BBB and since the anthocyanoside treatment was so general in effectiveness it may be anticipated that there would be a carry over from the rat to the human.

Another study on the permeability of blood vessels in general (not just the BBB) using rats showed "that previous treatment of animals with procyanidolic oligomers prevented the permeability increase produced by collagenase injection." ref 4 abstract "In animals pretreated with OPC the collagenase induced permeability increase is abolished. OPC protects the microvasuclar wall against the proteolytic attack." ref 4 fig. 1 caption This is important, because this is the very effect we want to achieve in our proposed treatment. I.e., by keeping the blood-brain barrier supplied with appropriate flavonoids, it is hoped that when something comes along that would normally have caused a breach and subsequent lesion, that instead the flavonoids will prevent damage from occurring. (Incidentally, OPC is the French equivalent of PCO).

Before discussing the manner in which flavonoids may potentially assert their effectiveness, we will first review some fundamentals. The BBB is a barrier which is caused by the capillary cells within the central nervous system (CNS) being bound together much more tightly than they are in the rest of the body. Because of the tightness, most substances are prevented from passing out of the capillary and into the CNS tissue proper. An extracellular matrix consisting of a number of proteins such as collagen and fibrin make up the matrix and function as a glue to hold the cells together.

Certain cells in the immune system produce enzymes which can dissolve the extracellular matrix. For most parts of the body this is useful in allowing white blood cells greater access to an area of infection. However, when these cells release their chemicals in the capillaries within the CNS, there is a resulting breakdown of the BBB, leading to an MS lesion, at least in certain individuals. One example of this process is with matrix metalloproteinases (MMPs), which, depending on their specific kind, can act on collagen, gelatin, fibronectin, and other components of the extracellular matrix. Of particular significance as we shall discuss shortly is that MMPs are not active in the form in which they are released, but must be converted to an active form by other enzymes. ref 5

There is another process at work which also has the potential to attack BBB integrity. During phagocytosis of target cells by phagocytes, free radicals are released. These free radicals are extremely active and can destroy many of the tissues they contact. This can be a useful feature when a phagocyte is trying to destroy an invading organism: "The superoxide ion and the derived oxygen free radicals are highly reactive and capable of destroying a variety of molecules. Their liberation during phagocytosis as potent bactericidal agents as well as their action in inflammatory events have been clearly demonstrated." ref 6 So, the generation of super oxides and free radicals are useful against infections. However, when the process takes place at the blood-brain barrier, such as takes place at the beginning of an MS lesion, it can be just one more attack on the integrity of the barrier.

I would now like to discuss three proposed mechanisms by which flavonoids might be effective in MS treatment.

1). Flavonoids have a tremendous affinity for joining themselves to enzymes. Thus, if they were to join themselves to either the MMPs or their activators, they might be able to block this process. This would then be a specific mechanism by which the BBB would be preserved and an impending MS lesion thwarted.

The following excerpts from an article on the flavonoids are relevant: "More recent studies indicate that certain flavonoids have rather extraordinary capacities to affect the activity of many enzyme systems.... It is also obvious that some of the enzyme systems affected by flavonoids are important in the regulation of secretory, contractile and motility processes, such as the release of inflammatory chemical mediators from mast cells, basophils, neutrophils, and macrophages." ref 7 In this article the author states that studies have clearly shown that certain flavonoids have an extraordinary capacity to affect the activity of the enzyme systems related to the ones we want to inhibit. The test results quoted above in ref 2 and ref 3 demonstrate these ideas on a practical basis. The mechanism we envision here is that when MMPs are released as discussed earlier, that the flavonoids would "disable" them before they joined with their activator enzyme. Thus, they would be rendered ineffective.

2) Flavonoids tend to become part of the cellular matrix, or at least adhere to it. This is particularly true of the PCOs. While in the matrix, they have receptor sites exposed which tend to grab onto the enzymes which would otherwise attack the matrix. Thus, they become "sacrificial lambs" so to speak in maintaining the integrity of the matrix. In an experiment it was "conclusively evidenced that PCOs bind to skin elastic fibres when injected intradermally into young rabbits. As a result, these elastic fibres were found more resistant to the hydrolytic action of porcine pancreatic elastase when injected to the same site. These in vivo studies further emphasized the potential effect of these compounds in preventing elastin degradation by elastase(s) as occurred in inflammatory processes." In the same article an in vito experiment is discussed where elastin is pretreated with PCO and as a result elastin becomes completely inhibited from dissolving the elastin: "The inhibitory effect of procyanidolic oligomers is even more pronounced with human leukocyte elastase. Pretreatment of elastin with this flavonoid completely inhibits the action of 10 micrograms of purified enzyme." "In the present work, we evidenced that flavonoids could efficiently and selectively bind to elastic fibres....Our results indicated that procyanidolic compounds bound on to insoluble elastin increased the non-productive binding sites of elastases(s)" ref 8 Although the medium here was elastin and not collagen or any of the other components of the BBB matrix, it is reasonable to anticipate that the effects would have been similar if collagen had been tested instead. Indeed, that is what was done in an experiment studied in the next paragraph.

3) Flavonoids are potent anti-oxidants. Free flavonoids (i.e., those not bound in the matrix) will rapidly destroy free radicals before the radicals do their damage. Although this is only a secondary issue, it could still be significant. In one experiment collagen taken from calf skin was exposed to super oxidants. It was found that the oxidants readily split the collagen into peptides of a relatively small size: "by use of a system generating oxygen free radicals in vitro, we were able to cleave fibrillar preparations of acid-soluble collagen into peptides of a relatively small size... We find here that the action of superoxide is even more complete and drastic than expected." ref 6 Thus, once inflammation starts in the blood brain barrier, any oxygen free radicals produced may be assumed to compound the attack on the BBB matrix, reinforcing the breakdown process. However, the authors showed that if the collagen were first soaked in anthocyanosides, allowing them to bind with the collagen, and next the collagen were rinsed so that there were no free anthocyanosides available, that the oxygen free radicals still had decreased ability to dissolve the collagen: "It was found that the anthocyanoside-treated fibrils do not contain any more free anthocyanoside and that the bound anthocyanosides do not exert a scavenging effect.... When the collagen fibrils have been pretreated by anthocyanosides prior to the exposure of the superoxide ion, the amount of solubilized peptides decreases..." Table 3 of the article discloses the degree of protection offered by various anthocyanosides and in various concentrations. Thus, the protection offered the collagen by pretreatment was due to action by anthocyanosides which had become bound to the matrix and were not simply floating freely. This confirms the process in step 2 above as well as showing the importance of anti-oxidants such as the flavonoids in bolstering the integrity of the collagen before it was attacked. Again, this is what we would like to accomplish in our efforts to prevent or reduce the number of new MS lesions by flavonoid treatment. It should also be pointed out that efforts to test the effects of super-oxide ions on the collagen but in the presence of free anthocyanosides were fruitless. The anthocyanosides neutralized the free radicals so quickly that the radicals never had any effect on the collagen: "It was not worthwhile to incubate collagen directly with both the superoxide ion generating system and the anthocyanoside preparations because the latter alone were found to directly inhibit the super-oxide forming system (Table 4)."

An extremely interesting experiment was performed with mice. Although MS does not naturally occur except with human beings, there is a disease called Experimental Autoimmune Encephalomyelitus (EAE) which behaves like MS. This diseased is induced experimentally in an animal such as a rat, mouse, or rabbit by artificially sensitizing the animal to proteins such as are found in its central nervous system. Once sensitized, the disease will progress in a fashion very similar to the naturally occurring human disease MS. In the experiment of interest, EAE was induced in mice. The progress of the disease was stopped by treating the mice with a particular drug. The following quotes are taken from the journal article recounting the experiment:

"Gelatinases, belonging to the matrix metalloproteases, contribute to tissue destruction in inflammatory demyelinating disorders of the central nervous system such as multiple sclerosis. We used experimental autoimmune encephalomyelitus (EAE) as an animal model to evaluate the effect of a hydroxamate matrix metalloprotease inhibitor (GM 6001) in inflammatory demyelination. ...results indicate that matrix metalloprotease inhibition can reverse ongoing EAE. This effect appears to be mediated mainly through restoration of the damaged blood-brain barrier in the inflammatory phase of the disease... ref 9

Notice that this experiment exactly parallels what we are trying to accomplish with our treatment of MS using flavonoids, except that GM 6001 is protein based and not a flavonoid. However, from the examples of the experimental results quoted above in ref 2 and ref 3 where blood-brain barrier integrity was restored or maintained by the use of anthocyanosides makes it appear that the results of this experiment might have been similar if appropriate anthocyanosides or PCOs had been used in place of GM 6001. Let's continue with a few more quotes from the same article:

"Since MMPs appear to play an important role in lesion development in inflammatory demyelinating diseases such as MS, inhibition of the activity of these enzymes might offer a new therapeutic approach in these disorders." Indeed, this is exactly what we are proposing, using flavonoids to inhibit the activity of the enzymes causing BBB breakdown.

"...the blood-brain barrier, which was significantly impaired in vehicle-treated animals, was restored in animals treated with the MMP inhibitor." The "vehicle-treated" animals are simply those in the control group who had similar injections to the test ones but without the GM 6001. So, effectively, the progress of EAE, an experimental equivalent of MS, was halted by use of a drug which inhibited MMP breakdown of the BBB. Again, this is exactly what we are proposing to do except by using flavonoids instead of protein-based inhibitors.

If we can indeed get the same results in MS using flavonoids as the experimenters did here in EAE using GM 6001, we will have a new, simple, cost-effective treatment for MS.

Reducing Inflammation: A Potential Side Benefit

In addition to strengthening the integrity of cell walls in general, including those of the blood-brain barrier, flavonoids have yet another potential value for the MS patient. Specifically, certain flavonoids have the potential ability to reduce inflammation. Since much of the damage caused to the nerve cells during an MS exacerbation is due to the inflammation, this would give hope that the treatment we will recommend to prevent or reduce the number of exacerbations might have as a possible side benefit that of reducing the severity of an exacerbation as well.

"The developing plaque of MS contains large numbers of macrophages (scavenger cells), which appear to destroy myelin by digesting its proteins and lipids. Inhibitors of the enzymes responsible for this digestion might reduce the myelin destruction or even interfere with movement of the macrophages into and through the tissues." "Improvements in MS are often dramatic. Much of the improvement is thought to be due to a subsidence of swelling and inflammation." "The principle drugs used to treat acute exacerbations are those having major antiinflamatory properties." "All of the above-mentioned drugs (i.e. certain drugs used to reduce inflammation) have possible serious side effects and should only be used under the supervision of a physician." ref 10 So, inflammation is a serious feature of an exacerbation and treatment of an acute attack typically centers on reducing inflammation with various drugs. Unfortunately, all of the drugs traditionally used have serious side effects.

"Anthocyanosides of natural origin have double-pharmacological action: (1) antidegenerative action ... (2) antiinflammatory action, which can be a direct reduction of capillary permeability or reduction of generation of inflammatory mediators. These common properties are considered to be due to their common chemical composition, having all of them a cumarin nucleus, while cell or tissue specificity of anthocyanosides comes from their variable side groups. The dual antidegenerative and antiinflammatory action promises wide use of anthocyanosides and related drugs..." ref 11 abstract Thus, even though our primary goal was to strengthen the blood-brain barrier, we have the added bonus of inadvertently, simultaneously reducing inflammation as well. Furthermore, we anticipate our proposed treatment to be WITHOUT side effects.

"Outstanding inflammatory effects are displayed by myricetin and delphindin, which contain vicinal hydroxy groups in ring B. The results confirm the importance of hydroxy group substitution in ring B." ("Delphindin" is an anthocyanidin.) ref 12 abstract Thus, anthocyanidins have "outstanding" antiinflammatory effects. (Note that an anthocyanoside is simply a compound whereby two sugar molecules are connected to an anthocyanidin).

Proposed Clinical Tests

In this section we shall derive a suggested treatment.

Fortunately, a person may significantly increase his anthocyanoside and/or proanthocyanidin intake quite inexpensively, typically less than one hundred dollars per month even doses at the therapeutic level. Everything needed to administer the test is readily available in a large supermarket or drugstore. Hence, the cost to clinically test the concepts of this paper would be minimal.

The International Federation of Multiple Sclerosis Societies published a book in which the following philosophy of treatment trial was expressed: "Another way of judging treatment efficacy in MS is to ask: Can this method prevent worsening? A completely effective treatment would prevent worsening in all cases, and probably produce improvements in most patients, and would be easy to recognize. A controlled trial of therapy would not be necessary. Unfortunately, no such agent has been discovered." ref 10 The least expensive clinical trial would be to test to see if certain flavonoids might indeed be this yet "undiscovered agent."

Anthocyanosides, procyanidins, and procyanidolic oligomers are all families of flavonoids. "There are over 500 varieties of flavonoids...with at least 20,760,000 members of the flavonoid class." ref 13 In a study using three different flavonoids to study their comparative abilities to protect against the degradation of calf skin under certain circumstances, it was confirmed that the three gave differing results. ref 6 Thus, we read that "It is essential to test a number of flavonoids of different chemical classes in various pharmacological screens of activity before a determination of activity, or lack thereof, can be made" ref 7

So, in considering a test of the effectiveness of flavonoids, we need to recognize that there are many, many different kinds of them and that they have differing effects on mammalian tissue. Furthermore, this is not a very well studied area. Thus, it is rational that our first attempts at clinical testing should simply supply a broad range of flavonoids from those classes whose history would lead us to anticipate favorable results. This is in opposition to an approach where one or two specific flavonoids would be used in a tightly controlled test, which might be appropriate once the initial basic information is available.

Furthermore, very little is known of proper dosage. However, it has been asserted that "no serious side effects have been observed with the use of flavonoids at moderate doses (<1 g/day/adult patient)." ref 13 Although flavonoids are quite widespread amongst vegetables and fruits and nuts, and we have no way of anticipating what a person's total flavonoid intake might be, we will propose a treatment based upon two principles. One is that we want to have as large a dose of the test flavonoids as possible, to make sure that their concentration is sufficient to be effective, and 2) if we set the limit of test flavonoid dosage to between 500 mg and 1,000 mg we will probably not have any significant side effects.

PCOs tend to be better anti-oxidants than anthocyanosides. ref 6 tables 3 and 4 On the other hand, anthocyanosides tend to be better antiinflammatory agents than PCOs. ref 12 For the time being we will simply suggest that treatment consist of both PCOs and anthocyanoside.

Blueberries tend to be between 0.1 and 0.2 percent anthocyanoside. ref 14 Using 0.15 % as an average computes to 720 mg in one pound of blueberries. Thus, fresh or frozen blueberries in an amount between one-half pound and one pound should be adequate for anthocyanoside intake. It is next recommended a PCO intake of 300 to 500 mg. total per day. The body disposes of excess flavonoids rather quickly, so the consumption of the above nutrients should be spread out reasonably evenly throughout one's waking hours.

As an intuitive starting point for lack of anything better, it would seem appropriate to initially start with an intake of closer to 1 gram per day of combined anthocyanosides and PCOs and then after a week or two tapering down to 500 grams per day. During an exacerbation for which symptom relief is desired, the intake would go back up to 1 gram per day with an emphasis on the anthocyanosides.

Anthocyanosides are also found in blackberries, cherries and other fruits. However, I have no information on the specific anthocyanosidic content of these fruits. In general, though, it would seem that a person would prefer the variety from mixing his intake of these three. Instead of fresh or frozen blueberries, one might want to take bilberry extract. Bilberry is simply European blueberry. The choice between fresh/frozen fruit versus an extract is probably mostly a matter of personal preference.

I found in a local health food store a PCO/Anthocyanoside source called Grape Complete, manufactured by C****** L***. Each capsule contains 50 mg of PCOs and 100 mg of anthocyanosides. 60 capsules came in a bottle for $23.00. Thus, 7 capsules a day would give us our 1 gram daily total target. With this product the fresh or frozen berries would not be needed. The monthly cost for this would be about $84.00. Even with vitamin C supplements added (see discussion below) the cost is less than one hundred dollars per month during the intensive portion and would probably be closer to fifty or sixty dollars a month during normal situations. I do not know the relative quality of this or any other brand; therefore I cannot make any meaningful product recommendations.

I would like to see two different groups of tests be performed to determine the general effectiveness of flavonoids in the treatment of multiple sclerosis.

The first group of tests would be with those who have rapidly advancing MS. This would be a small group, perhaps six to twelve in number. The test would last three to six months. Three MRI scans of all participating members would be taken. The first would be at the outset of the test, the second after one month so that a reference would be provided after the anthocyanosides would have had opportunity to stabilize the development of new lesions, and then the third at the end of the test period. At the conclusion of tests the second and third scans would be compared to see how effective the anthocyanosides were in dealing with the disease.

A second group of tests would be made with patients having the more traditional remission-relapse form of the disease. This group would need to be much larger, probably at least fifty if not more. This is because two-thirds of patients having recent exacerbations will show subsequent improvement, albeit ultimately only temporary, even without treatment. Hence, for anthocyanoside to be considered effective in this group the size of the group must be large enough that statistical fluctuations will not color the results. With a group of fifty patients, we would expect there to be only sixteen or so who did not improve even without taking any kind of treatment. If, in a group of fifty patients there were improvements in all but five or six patients, this would be considered significant. If the group size were much less than fifty, it would be difficult to assess the significance of the results.

In evaluating the results of the tests one should bear in mind that the flavonoids have a possible dual role, that of reducing the number and frequency of exacerbations and also that of alleviating some of the symptoms during an exacerbation. Those making evaluations need to keep this perspective in view.

Vitamin C seems to work cooperatively with anthocyanosides, ref 15 p. 367 Therefore, I would also recommend that a 100 mg vitamin C tablet be taken three times a day, simultaneously with the other nutrients.

Depending on the results of these tests, future test plans can be established. The advantages of the proposed treatment in this article are 1) they are consistent with the concepts behind the latest theories of MS 2) they are relatively inexpensive and readily available 3) they are potentially good for a person's health in general, independent of their effectiveness with MS (unlike many of the immuno-suppression and antiinflammatory approaches used historically and currently).

Please note that these recommendations are merely suggestions for clinical tests administered by qualified researchers. They are not intended for self-treatment by an individual apart from the direction and supervision of his personal physician.

Note added 3/21/97: Over the past nine months anecdotal results of people trying some of these ideas have accumulated. A representative sample is posted at http://www.innercite.com/~tstout/ms/anec.shtml.

Ref 1

M Lai, et al, "A preliminary study into the sensitivity of disease activity detection by serial weekly magnetic resonance imaging in multiple sclerosis." Journal of Neurology, Neurosurgery, & Psychiatry. (1996) 60 n. 3: 339-341.

Ref 2

A. M. et al. "Action of anthocyanosides of Vaccinium Myrtillis on the Permeability of the Blood Brain Barrier." Journal of Medicine. (1977) 8 n. 5: 321-332.

Ref 3

Z. Detre et al. "Studies on Vascular Permeability in Hypertension: Action of Anthocyanosides." Clin. Physiol. Biochem. (1986) N.4: 143-149

Ref 4

L. , et al, "Action Des Oligomeres Procyanidoliques Sur La Permeabilite De La Paroi Vasculaire. Etude Par Morphologie Qunatitative." Path Biol, (1990), 38, no.6: 608-616.

Ref 5

A. Maeda et al. "Matrix Metalloproteinases in the normal Human Central Nervous System, Microglial Nodules, and Multiple Sclerosis Lesions." Journal of Neuropathology and Experimental Neurology, (1996), 55 n. 3: 300-309

Ref 6

Claude Monboisse, et al. "Non-Enzymatic Degradation of Acid-Soluble Calf-Skin Collagen by Superoxide Ion: Protective Effect of Flavonoids". Biocemical Pharmacology, (1983), 32 n. 1: 53-58.

Ref 7

Elliot Middleton, Jr., "The Flavonoids", Trends in Pharmacological Sciences. (August 1984), 5: 335-338

Ref 8

J.M. Tixier, et al. "Evidence By In Vivo and In Vitro Studies That Binding of Pycnogenols to Elastin Affects its Rate of Degradation by Elastases. Biochemical Pharmacology (1984), 33, N. 24: 3933-3939

Ref 9

K. Gijbels et al. "Reversal of Experimental Autoimmune Encephalomyelitius with a Hydroxamate Inhibitor of Matrix Metalloproteases." The Journal of Clinical Investigation, (1994), 94: 2177-2182.

Ref 10

W.A. Sibley and the Therapeutic Claims Committee of the International Federation of Multiple Sclerosis Societies, Therapeutic Claims in Multiple Sclerosis, 3rd Edition. 1992.

Ref 11

J.M. Feher et al. "Chorioretinal Myopic Degeneration: Treatment with Anthacyanosides." Bolletino di Oculistica. (1990), 69 n. 5: 909-922

Ref 12

M. Gabor et al. "Effect of Benzopyrone Derivatives on Simultaneously Induced Croton Oil Ear Oedema And Canageenan Paw Oedema in Rats." Acta Physiologica Hungarica. (1991), 77, n.3-4: 197-208

Ref 13

B. Havsteen. "Commentary. Flavonoids, A Class of natural Products of High Pharmacological Potency". Biochemical Pharmacology. (1983), 32 n. 7: 1141-1148.

Ref 14

W. Lenartowicz et al, "The Quality of Highbush Blueberry Fruit: PI. Fresh Fruit Quality of Six Highbush Blueberry Cultivars and Their Suitability For Freezing." Fruit Science Reports (Skierniewice). (1990), 17 n.2: 77-86

Ref 15

M. Gabor. "Pharmacologic Effects of Flavonoids on Blood Vessels. Angiologica, (1972), 9: 355-374 (223-242)

© 1996 by R. Stout

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