Medicine:

Advancements in Neurology:
Innovations in Stroke Treatment

by Dr. Hiroshi Morooka

Okayama, Japan

 

Editor’s note: This paper was originally presented at the 2005 International BWW Conference in Las Vegas, the United States, on August 10, 2005. Dr. Morooka has also presented papers at the 2004 BWW Conference in Xi’an, China and the 2003 BWW Conference in Antequera, Spain.

 

We presented a paper at the 2003 BWW Congress at Antequera: The sympathetic nervous system is especially involved in response to many stress. This sympathetic stimulation to various stress induces activation of procoagulant mechanism on the systematic level. In other words, mental reaction to stress could be responsible for human ageing.

　

In ischemic process, platelets are activated by agonists such as norepinephrine ( NE ),

epinephrine, arachidonic acid (AA), thromboxane A_２(TXA_２), serotonin, collagen, adenosine diphosphate (ADP),thrombin and H_２O_２(reactive oxygen radical).

 

Foremost among the platelet-derived humoral mediators of tissue injury were the prostaglandins (AA,PGF_２α), the endoperoxides (PGH_２), and TXA_２. 　TXA_２and to a lesser extent, PGF_２α and PGH_２are potent vasoconstrictors and can reduce blood flow to vital vascular beds. More over, these agents are prothrombotic by virtue of potently stimulating platelet aggregation. 

 

Platelets are the primary source of TXA_２. Release of TXA_２ aggregate other platelets and stimulates their release of additional TXA_２. These platelet propagate process are all considered to be major factors, contributing to direct ischemic injury as occurs in myocardial and cerebral ischemia.

 

Cellular mechanism of arachidonic acid produces not only TXA_２, but also leukotrienes,   a group of lipid mediators that initiate, aggravate, or sustain vascular inflammatory disorders.

 

The cysteinyl leukotrienes ( LTC_４,LTD_４,and LTE_４) constrict vessels and promote vascular permeability. We studied these harmful leukotriens and the role of platelet-leukocyte interaction in hemostasis, thrombosis and atherosclerosis(Fig.1).

Fig.1.　Reaction catalyzed by 5-lipoxygenase

 

Platelet aggregation curve was obtained by the change of light transmission, using NBS HEMA TRACER. 150 patients with cerebral infarction were study participants ( age, 66.96± 9.43years and 71.04± 8.92years; 66 men, 84 women).  Platelets rich plasma (PRP) was separated by the centrifugation (800rpm, 15min) of the mixtures, containing 3.8% citric acid / blood (1/9).

 

Platelet aggregation curves induced by ADP (1.5μM) were divided into 3 types. Type 1: Maximum amplitude of aggregation was less than 40% with dissociated curve from aggregation. Well–treated patients had the type 1 . Type 2: Maximum amplitude was between less than 60% and more than 40%. Type 3: Maximum　amplitude was more than 60% with secondary aggregation. Patients with cerebral infarction at acute stage had the type 3.                                                                

 

Patients exposed to stress conditions, including hypertension, smoking, cold temperature, low atmospheric pressure, pain, anxiety, anger, and/or fear, also revealed the type 3 (Fig. 2).

Fig. 2.

Leukocyte-mediated potentiation of platelet aggregation

　Polymorphonuclear (PMN) leukocytes from human blood were obtained by the Hypaque-Ficoll method.^１ PMN leukocytes were suspended in the SOLITA^®-T No.3

solution, composed of (in w/v %) 0.09NaCl, 0.149KCl, 0.224C_３H_５NaO_３ and 4.3%C₆H_１２ O_６.  PMN leukocytes were added to PRP.

Platelet　aggregatory response to various agonists in PLATS/PMN (100/1) were more augmented than that in platelet alone(Fig.3).

Fig.3. Leukocytes mediated potentiation of platelet aggregation induced by various agonists such as ADP, collagen, thrombin, AA and phorbol myristate acetate (PMA). 1: Platelets having type 3-aggregation. 2: Platelets having type1-aggregation. Platelet aggregation in PLATS/PMN was potentiated as compared with that in PLATS alone.  

 

Expression of p-selectin in activated platelets

Surface expression of p-selectin is a marker for α-granule secretion in platelet and mediates leukocyte adhesion to activated platelets and endothelial cells.^２ Activate platelets produce p-selectin which gathers and activates leukocyte. Leucocyte tethering and rolling has been demonstrated to primarily be mediated by the selectin family of adhesion molecules. Plasma p-selectin was measured using a high-sensitivity enzyme-linked immunosorbent assay (ELISA, Bender Med.Systems).

 

P-selectin levels in the plasma containing platelets with type 3-aggregation (type 3-platelets) after activation by various agonists were higher than that of control  (Fig.4).

Fig.4. Plasma p-selectin levels after platelet activation induced by various agonists.

●: platelets having type 3-aggregation (type 3-platelet),○: platelets having type 1-aggregation (type 1-platelet).

 

 

Leukotriene B_４production in PMN leukocytes

Leukotrienes were measured using EIA method ( Cayman Chem. Com.).

Leukotriene B_４is a potent stimulus for PMN leucocytes, eliciting increases in migration, adherence, degranulation, superoxide production and cytotoxicity.

Levels of leukotriene B_４in the plasma containing type 3-platelets mixed with PMN leukocyte after activation by various agonists, were significantly greater than that in Type 3- platelets alone　(Fig.5).

 

.

Fig.5. Levels of the plasma leukotriene B_４after activation of PMN leukocytes. ●: PMN and PLATS,○: PMN alone.

 

Increased levels of the plasma leukotriene B_４, containing type-3 platelets and PMN leukocytes, occurred after activation by various agonists such as ADP, collagen, thrombin, AA or PMA , because platelet-derived AA may serve as a precursor for the 5-lipoxygenase enzyme in the PMN leukocytes.^３

 

Cysteinyl leukotrienes production in interactions between activated platelet and PMN leukocytes..

Leukocytes contain phospholipase A_２and 5-lipoxygenase.   Thus they can generate LTA_４.In contrast, other blood cell, such as erythrocytes and platelets or vascular endothelial cells contain LTA_４hydrolase  or LTC_４synthase, but they lack 5-lipoxygenase and cannot generate their own LTA_４. Activated platelet may receive  LTA_４ from PMN leukocyte ,using  transcellular biosynthesis, and produce LTC_４.^４  LTC_４and its metabolites LTD_４,LTE_４, are potent mediators of vasoconstriction and increase vascular permeability.

 

 

The plasma LTB_４ and LTE_４, containing type3-platelets, revealed high levels as compared with those containing type 1-platelets. Patients with cerebral infarction at acute stage had type-3 platelets (Fig.6).

Fig.6.  Enhanced production of leukotrienes in patients with cerebral infarction.

 

Conclusions

A recent study has shown that C-reactive protein or numbers of leukocytes, a marker of inflammation, was a story predictor of cardiovascular events. It has been postulated for more than a century that infection may be responsible for atherosclerosis.

Chlamydia pneumonia, Cytomegalovirus, Helicobacter pylori, and periodontal infections may be associated with increased risk of carotid atherosclerosis or stroke.^5,6,7

 

These organisms were localized with plaque ulceration and thrombosis. Chronic damaged endothelium may disclose collagen or tissue factors. These substances activate platelet aggregation. Activated leukocyte produce H_２O_２, leading platelet aggregation.

More over　activated  leukocytes  produce LTA_４and LTB_４in platelet-leukocyte interaction. Platelet don’t have lipoxigenase, so platelets use LTA_４produced by leukocytes and generate LTC_４, LTD_４and LTE_４.These leukotriene may give damage to microcirculation.

 

Our previous experiments have shown that histamine release is accelerated by free radicals, which may contribute to the increase of macromolecular permeability of the blood brain barrier and to vasogenic edema.^８ 

 

To treat stroke appropriately, we may use cyclooxygenase inhibitor for TXA_２ and also inhibitory drugs for leukotrienes production.                                       

 

The ways to keep our healthy condition are as follows: 1. We must have mutual relationship and don’t reveal offensive attitude against stress; 2. We should not take much foods , because obesity also cause increase sympathetic stimulation. We should reduce our body weight under the moderate excise; 3. We should take more antioxidant food, containing vitamin E, vitamin C and polyphenols; 4. We have to keep our body away from infection. Antibiotics should be used for suspected infection.

 

Finally, we should respect individual life and make effort to enhance perpetual peace of all the world with brotherly love.

 

 

References

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2.      Palabrica T, Lobb R, Furie BC,　Aronoviez　M,Benjamin　C,　Hsu YM、Sajer　SA, Furie　B.　 Leucocyte accumulation promoting fibrin deposition is mediated in vivo by p-selectin on adherent platelets. Nature 1992; 359:848-850.

3.      Palmantier R, Borgeat P. Thrombin-activated platelets promote leukotriene B_４synthesis in Polymorphonuclear leucocytes stimulated by physiological agonists. Br. J. Pharmacol. 1991; 103:1909-1916.

4.      Maugeri N, Evangelista V, Celardo A, Dell’Elba G, Martelli N, Piccardoni P, de Gaetano G, Cerletti C. Polymorphonuclear leukocyte-platelet interaction; Role of p-selectin in thromboxane B_２and leukotriene C_４cooperative synthesis. Thromb. Haemost. 1994; 72:450-456.

5.      Cook PJ, Honeybourne D, Lip GY,　Beevers　DG, Wise　R,　Davies　P. Chlamydia pneumoniae antibody titers are significantly associated with acute stroke and transient cerebral ischemia: the West Birmingham Stroke Project. Stroke. 1998; 29:404-410.

6.      Chiu B. Multiple inferctions in carotid atherosclerotic plaques. Am Heart J. 1999; 138 (5Pe2):S 534-S 536

7.      Farsac B, Yildirir A, Akyon Y,　Pinar　A,　Oc M、Boke　E、Kes　S,　Tokgozoglu　L.  Detection of Chlamidia pneumoniae and Helicobacter pylori DNA in human atherosclerotic plaques by PCR. J.Clin. Microbiol. 2000; 38:4408-4411.

8.       Morooka H, Sasayama H, Sakai K, Namba S, Nishimoto A. The role of catecholamine-induced lipid peroxidation and histamine in ischemic brain edema. In: Hoff JT, Betz AL (eds) Intracranial pressure VII. Springer, Berlin Heidelberg, New York, 1989:818-820