Paula, I will be seeing the London Research group on Monday, and am collecting all the evidence in one place, to show them. When I have fginished it I will post the full report here.
In the meantime, here is an interesting followe on from the Gulf War story.
Ron

STRESS AND THE PERMEABILITY OF THE BLOOD-BRAIN BARRIER

Category: Neurochemistry
Term Paper Code: 146

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The blood-brain barrier (BBB) is the biological barrier that protects the brain by regulating the flow of numerous substances into the brain. These substances can range from the rapidly circulating ions to neurotransmitters, growth factors, bacteria and their toxins, and other substances that can affect the central nervous system. What do go through the BBB are the small hydrophobic or lipophilic, non-ionic, and non-polar molecules, because they can cross or diffuse into the membrane more easily. The BBB consists of a continuous layer of endothelial cells that are joined by tight junctions with very high electrical resistance (Hanin 1996). This concept of the BBB was first introduced by a famous bacteriologist named Paul Ehrlich, when he demonstrated that intravenously infected dyes stained most organs in the body, but the brain remained unstained (Hanin 1996).

However, the BBB is not as a rigid, non-flexible barrier like the Wall Berlin as what people usually associate the word “barrier” with. It is the permeability of the BBB that governs the “traffic flow” of the substances that go in and out of the brain, and therefore, there is no definite set of substances that always can or cannot cross the BBB. A very interesting phenomenon was introduced in MCB 165 course regarding this permeability of the BBB. Citing the work of Friedman (1996), Prof. Presti suggested that the soldiers who had participated in the Gulf War might have had some penetration of the substance pyridostigmine into their brains, when it was administered to the soldiers as a pretreatment against soman poisoning. Soman is an organophsphate nerve gas routinely used in Western armies, which can inhibit acetylcholinesterase in the peripheral nervous system to cause abnormal parasympathetic functions. Pyridostigmine also inhibits acetylcholinesterase, but due to its quaternary ammonium group, it cannot cross the BBB under the normal condition. What that means is that, at best, it can only exert its effect on the peripheral nervous system in the forms of diarrhea, excess sweating and increased salivation. This is the very reason why pyridostigmine is commonly used as a pretreatment against acetylcholinesterase-inhibiting nerve gases. Also, thousands of people are taking pyridostigmine safely everyday as a treatment for chronic neuromusclular disorder called myasthenia gravis, because the side-effect of pyridostigmine is minimal compared to the severe symptoms that the disease causes. However, the findings of the Israeli scholars, Sharabi et al. (1991), report a threefold increase in the frequency of central nervous system symptoms (headaches, insomnia, drowsiness, nervousness, unfocused attention, and impaired capacity to conduct simple calculations) following pyridostigmine ingestion by 213 Israeli soldiers treated during the Persian Gulf War. This clearly indicates that there must have been some penetration of pyridostigmine through the BBB. Friedman (1996) suggests a promising explanation to this possible penetration by demonstrating that stress causes changes in the permeability of the BBB. Using mice in his experiment, Friedman found out that those mice that were subject to forced swimming (a known stressed condition) reduced the pyridostigmine dose required to inhibit mouse brain acetylcholinesterase activity by 50% to less than 1/100th of the usual dose. Hence, there seems to be a significant correlation between stress and the permeability of pyridostigmine into the central nervous system. From this, he concluded that peripherally acting drugs administered under stress may reach cross the BBB and affect centrally controlled functions.

I was very intrigued by this phenomenon when I read the article in the reader, and when it was presented in the lecture. It is in line with the current trend of various findings which suggest that stress is not an arbitrary, implicit, or exclusively emotional factor anymore, but a very important part of understanding human physiology. I have always wanted to see more research literature that makes some comprehensive approach to link what is commonly referred to as being “not scientific,” “taboo,” “subjective,” or “implicit” with empirical explanations. Saying “well, that’s just too subtle and since we cannot measure it, I won’t even make any attempt to describe it in terms of science- let’s forget it!” to subject matters like mind of spirituality is, I believe, just being idle on there jobs.



I, therefore, searched through literatures to find more related researches done on the topic of the relationship between stress and BBB. I have found many, and they all seem to support Friedman’s conclusion. However, due to the limited access to many of the sources, I was able to get the full text for only some of these articles. These are:


1. Hanin, I. The Gulf War, stress and a leaky blood-brain barrier. Nature Medicine. 2, 1307-1308 (1996).


2. Azevedo, I. & Sarmento, A. Stress and the blood brain barrier. Nature Medicine. 3, 253 (1997).


3. Sarmento, A., Arbino-Teizeira A. & Azevedo, I. Amiriptyline-induced morphological alterations of the rat blood-brain barrier. European Jounal of Pharmacology. 176, 69-74 (1990).


4. Sharma, H.S., et al. Probable involvement of serotonin in the increased permeability of the blood-brain barrier by forced swimming. An experimental study using Evans blue and I-sodium tracers in the rat. Behavioral Brain Research. 72, 189-196 (1996).


All of these four articles have the same theme of the correlation between stress and increased permeability of the BBB, but each shed light on different places. Now, I will go through each of them in more detail, and combine them at the end to draw some meaningful conclusion.


1. The Gulf War, stress and a leaky blood-brain barrier (Hanin 1996).

This article was in the “News & Views” section of the Nature Medicine, and thus very overviewing and commentary in its nature. It briefs over the history of the discovery of the BBB, its structures, functions, and current pharmacological applications on it. Then, it introduces the idea of possible penetration of pyridostigmine into the soldiers in the Gulf War due to the altered permeability of their BBB, along with introduction of Friedman’s work and related articles for this idea. It lists out the possible causes of stress that ultimately results in changes in the BBB, such as acute immobilization stress in rats, cold or isolation exposure in mice, and exposure of rats to conditions of acute as well as chronic summer heat. Each of these examples represents a separate work done by other researchers, and thus, this particular article helped me very much in that it was almost a “guide” to this whole research for me. The conclusion that this author drew from his review on Friedman’s work and others was that we must look at the BBB from a different perspective than the classical textbook view, which teaches that the BBB is a very effective barrier to penetration of undesirable molecules into the brain. This is very much in alignment with my opinion stated initially, that the BBB should not be described as something “rigid” in its nature. There is a constant change in its permeability and flow, depending on such common stimuli as stress in human experience. The “rigid” and “effective” model of the BBB is certainly true when everything is under normal conditions, but once stress becomes a part of the picture, this premise is very much likely to fall apart. Therefore, modifications on the descriptions of the BBB are required for a better and precise understanding of this important brain structure. This basically represents the conclusion of this article.



2. Stress and the blood brain barrier (Azevedo & Sarmento 1997).

This article is yet another commentary one, and actually a follow-up on the previous article by Hanin (1996). Azevedo et al. published this article in the “Letters to the Editor” section of the Nature Medicine in March 1997 issue. These authors acknowledge Friedman’s work, but suggest that Hanin (1996) did not discuss the cause of the increased BBB permeability associated with stress. They had actually performed several studies aimed at clarifying the modulatory effects of central adrenergic mechanisms on the BBB and had reported their results as early as 1990. The topics of their past reports were: an amitriptyline-induced increase in brain capillary endothelial cell pinocytosis (European Journal of Medicine, 176, 69-74,1990); an increase in BBB permeability following intracerebroventricualr administration of catecholamines (Nunyn-Schmiedeberg’s Arch. Pharmacol. 343, 633-637, 1991); and an increase in permeability after electrical stimulation of locus coeruleus in rats (Acta Neurocirurgica. 127, 215-219, 1994). They all speculate that the possible cause of the change in the permeability of the BBB is related to some exposure of the microvessel endothelial cell monolayer (i.e. the BBB) to adrenergic drugs. Since these catecholamines are well-known stress hormones, these authors suggest that their findings shed some light on the mechanism implicated in stress-induced increased permeability of the BBB. The hypothesis of these authors seemed very interesting in this regard-that it is actually the catecholamine activities which causes the profound change in BBB permeability, -but I could only get one article of the many mentioned above. I will delve more into this “catecholamine hypothesis” as it appears in the next article.


3. Amiriptyline-induced morphological alterations of the rat blood-brain barrier (Sarmento et al. 1990).



This is relatively an old article, and yet no other author offered as a detailed report as this one did within the last 9 years in terms of the “catecholamine hypothesis”. The experiment in this article used amitriptyline as a central molecule that causes an increase in the permeability of the BBB. Since morphological basis of this phenomenon has not been studied, the authors attempted to give possible explanations by emphasizing that amitriptyline is an inhibitor of the neuronal uptake of norepinephrine (NE, a kind of catecholamine), which in turn had been proven to influence pinocytosis in peripheral canine blood vessels. It is well known that the low pinocytotic activity in cerebral capillary endothelial cells takes up one important feature of the BBB. Therefore, the authors targeted the effects of amitriptyline on pinocytotic activity of the brain microvessels as a promising morphological basis. In other words, the authors explain the phenomenon of increase of BBB permeability by amitriptyline through the chain reactions of different steps involved, i.e. amitriptyline, NE, and the pinocytosis. Amitriptyline would cause a decrease in the neuronal reuptake of NE, which in turn would cause increase in the pinocytotic activities, which then would result in creation of vesicles in the cerebral capillary endothelial cells. These vesicles would offer the morphological basis for the increased BBB permeability.

In this study, amitriptyline was injected to rats and the parietal cortex of control and treated animals was prepared for ultrastructural study. Then, the pinocytotic vesicles in endothelial cells were counted. The results showed that amitriptyline significantly increased the density of pinocytotic vesicles in capillary endothelial cells. Even without a dose-response curve, the authors believed that the parallelism between the effects of amitriptyline (given at a specified dose and time schedule), on both the permeability of BBB and pinocytosis of brain capillaries suggests a relationship between these two effects.

The framework of these authors came largely from the work of Preskorn et al. (1982) which showed that tricyclic-antidepressants, e.g. amitriptyine, increased the permeability of the BBB to water. This effect was attributed to an increase in central adrenergic activity due to the inhibition of the neuronal re-uptake of NE by these drugs, and therefore Preskorn et al. suggested that the adrenergic innervation of brain capillaries might control BBB permeability.

The mechanism of the effect of amitriptyline on pinocytosis is still unknown because amitriptyline involves several actions: serotoninergic, anticholinergic and histaminergic effects, even on top of the aforementioned inhibiting effect on the neuronal uptake of NE. However, the authors ruled out everything but the last effect by citing the work done by Preskorn et al. (1982) again. Preskorn et al. (1982) had demonstrated that the effect of amitriptyline on BBB permeability was prevented by previous central adrenergic denervation with 6-hydroxydopamine or by pretreating with an alpha blocker. They also claimed that they ruled out the possible serotoninergic, anticholinergic or antihistaminergic effects in some way that was not mentioned clearly. However, it must have been done with a similar logic. To further support their “catecholamine hypothesis”, Sarmento et al. also mentioned the work of Raichle et al.(1975), which showed that the brain vascular permeability to water increases when noradrenergic cell bodies in the locus coeruleus are directly stimulated with carbachol.

In summary, the authors of this study showed that it must be the vesiclization of the BBB which is caused by the increased pinocytotoxic effects of amitriptyline, which results in increased permeability of the BBB.



4. Probable involvement of serotonin in the increased permeability of the blood-brain barrier by forced swimming. An experimental study using Evans blue and I-sodium tracers in the rat (Sharma et al. 1996).



This article approaches the general theme of increased BBB permeability in a different way. Although the serotoninergic effect of amitriptyline, the substance that was central to the previous study by Sarmento et al., was ruled out, serotonin (5-HT) from other route is viewed as another promising agent of the action in this particular study. Citing the work done by Boullin (1978) and Essman (1978), the authors state that there are indications that altered serotonin metabolism occurs in a wide variety of neurological decreases which may selectively increase the permeability of the BBB. Then the authors mention the “Forced Swimming (FS)” protocol which is well known to be a severe stressful condition in which alteration of serotonin metabolism occurs. Therefore, they come to speculate the possibility of the altered metabolism of 5–HT being able to increase the permeability of the BBB in FS. Since it has been shown by many researchers that 5-HT is involved in various physiological mechanisms of stress, that the metabolism of 5-HT is altered following stressful conditions, and thus, that altered metabolism of 5-HT actually occurs in FS, the authors attempt to examine the status of BBB permeability and serotonin metabolism following FS in rats of different ages. Moreover, the authors attempt to account for the implication of prostaglandins (PGs) as a first mediator of stress response and what actually may stimulate 5-HT synthesis in the brain, by inhibiting 5-HT synthesis with indomethacin in this influence of PGs on the BBB permeability and 5-HT level in FS.

Conscious young rats were subject to continuous FS for 30 minutes, which is relative a short-term stress. Then the status of the permeability of the BBB was examined by using Evans blue albumin (EBA) and 131I-sodium in brain regions. These tracers bind to serum albumin in vivo, and thus, extravasation of these tracers into the brain represents mainly the leakage of tracer-protein complex. The 5-HT in plasma and brain was measured by using a sensitive fluorometric assay. The results showed that the BB permeability to EBA and 131I-sodium was markedly increased in animals after 30 min FS. This increase, however, was absent in animals which got a rest period of 120 min after the same length of stress (30 min), indicating that FS-induced increase in BBB permeability was reversible. Also, there was no significant increase in animals subjected with 5 min and 15 min period of FS, either, indicating that the duration of FS is an important factor in inducing the breakdown of BBB permeability. The 5-HT levels in the plasma and brain of the rats showed similar trend across the four groups of rats, i.e. the group subjected to 30 min FS showed the highest increase and no increase in 5 min nor 15 min FS. Therefore, it appears that serotonin may play an important role in the probable mechanisms of increased permeability following FS. Serotonin is actually a potent vasoactive agent which is known to modify the permeability of the BBB by a specific receptor mediated mechanism. In this study, effects of other selective drugs on these parameters were also examined, to see the importance of 5-HT in these various receptors. First of all, increased permeability of the BBB following FS was prevented, when blockade of increased 5-HT level in plasma and brain as well as antagonism of 5-HT2 receptors in spite of high plasma and brain 5-HT levels happened by pretreating the animals with p-CPA (a 5-HT synthesis inhibitor), Cyproheptadine (a 5-HT2 receptor antagonist), and Ketanserin (a specific 5-HT2 receptor antagonist). These suggest that the binding of serotonin to 5-HT2 receptors is important in the breakdown of the BBB permeability that follows FS. Secondly, increased vascular permeability was completely prevented, when indomethacin (a potent PG synthesis inhibitor) in a concentration high enough to block the PG synthesis in the cerebral vessels was pretreated in the animals. The fact that the blockade of the BBB permeability in FS by indomethacin suggests that there are PGs involved in the permeability. This is in line with the findings which show that PG is a first mediator of stress and the inhibition of their release with indomethacin will prevent the stress response in animals. Since PGs are also known as stimulator of 5-HT synthesis, it seems likely that indomethacin prevents the release of PGs in response to FS, which in turn prevents the increase of 5-HT in plasma and brain.

In summary, subjection of young animals to 30 min FS induces an early release and accumulation of serotonin in plasma. The increased level of 5-HT then acts on 5-HT2 receptors located on cerebral vessels. Once bound to 5-HT2 receptors, stimulation of the synthesis of PGs begins. PGs in turn may further stimulate the accumulation of cAMP. This accumulation of cAMP in cerebral vessels will enhance vesicular transport resulting in extravasation of tracers across cerebral vessels.


As demonstrated clearly by the four articles reviewed so far, the phenomenon of increased BBB permeability under stress can be resulting from many different domains of the brain chemistry. If we take all the possible explanations mentioned together, we get something like the following:

1. Stress involves immediate neurochemicals like catecholamines (norepinephrine or epinephrine), amitriptylines, prostaglandins, and serotonin, which further affect the manifestation of the increased BBB permeability through various routes.

2. The vesiclization of the BBB is caused by the increased pinocytotoxic effects of amitriptyline, which in turn is caused by the inhibition of noradrenaline uptake. This results in increased permeability of the BBB.

3. When accumulation of serotonin in plasma occurs, the increased level of 5-HT then binds to 5-HT2 receptors located on cerebral vessels, which, in turn, stimulate the synthesis of PGs. PGs in turn may further stimulate the accumulation of cAMP, and once accumulated, cAMP in cerebral vessels enhances vesicular transport resulting in increased permeability of the BBB.



Therefore, there seems to be two main explanations to this phenomenon: “catecholamine hypothesis” vs. “5-HT hypothesis”. Each has merits in its own explanation, and at this point, we cannot determine which one is the correct one. Perhaps both of these are happening when the BBB permeability changes under stress, indicating that some kind of network is there between the two pathways. Or, it may be that neither of these actually offers a complete explanation to the phenomenon, i.e. we have to include some other factors in. This is highly likely to be the case, since there are other findings that suggest completely new approaches to this same phenomenon. One internet site actually suggests that the pyridostigmine that was used in Gulf War was able to result in nerve damages because it was accidentally mixed in combination with pesticides commonly used around the area (WWW1). Hanin (1996) and many other researchers suggest that the increased penetration of drugs into the BBB may be exacerbated by high temperature, cold or isolated exposure, and/or mood as well. There are also other findings which suggest that opioid receptor antagonists attenuate heat stress-induced reduction in cerebral blood flow, increased BBB permeability, vasogenic edema and cell changes in the rat (Sharma et al. 1997). Yet another agent introduced in other study was the possible neurotoxin glutamate (Skultetyova et al. 1998). Therefore, no single explanation so far has been successful in fully accounting for the phenomenon in question.

In conclusion, there appears to be a lot more research required, in order to provide a complete explanation to the phenomenon of the increased BBB permeability under stress, as all the authors in the aforementioned studies unanimously suggested. The complete explanation would have to include the exact mechanisms in the phenomenon. Hence, it appears that only a comprehensive links amongst the currently suggested and yet-to-come findings can promise a concrete explanation.

References

WWW1: http://vergemagazine.com/context/fea...lfvaccine.html

Azevedo, I. & Sarmento, A. Stress and the blood brain barrier. Nature Medicine. 3, 253 (1997).

Boullin, D., Serotonin in Mental Abnormalities, Wiley, Checester, 1978, pp. 1-340.


Essman, W., Serotonin in Health and Disease, Vols, I-IV, Spectrum, New York, 1978.


Friedman, A., Kaufer, D., Shemer, J., Hendler, I., Soreq, H. & Tur-Kaspa, I. Pyridostigmine brain penetration under stress enhances neuronal excitability and induces early immediate transcriptional response. Nature Medicine. 2, 1382-1385 (1996).


Hanin, I. The Gulf War, stress and a leaky blood-brain barrier. Nature Medicine. 2, 1307-1308 (1996).


Sarmento, A., Arbino-Teizeira A. & Azevedo, I. Amiriptyline-induced morphological alterations of the rat blood-brain barrier. European Jounal of Pharmacology. 176, 69-74 (1990).Sharabi, Y. et al. Survey of symptoms following intake of pyridostigmine during the Persian Gulf War. Israel Journal of Medical Science. 27, 656-658 (1991).


Sharma, H.S., Westman, J., Cervos-Navarro, J., Dey, P.K. & Nyberg, F. Probable involvement of serotonin in the increased permeability of the blood-brain barrier by forced swimming. An experimental study using Evans blue and I-sodium tracers in the rat. Behavioral Brain Research. 72, 189-196 (1996).


Sharma, H.S., et al. Opioid Receptor Antagonists Attenuate Heat Stress-Induced Reduction in Cerebral Blood Flow, Increased Blood-Brain Barrier Permeability, Vasogenic Edema and Cell Changes in the Rat. Annals New York Academy of Sciences. 813O: 559-71 (1997).


Skultetyova I., Tokarev D. & Jezova, D. Stress-Induced Increase in Blood-Brain Barrier Permeability in control and Monosodium Glutamate-Treated Rats. Brain Research Bulletin. 45, 175-178 (1998).

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