Drinking game of the brain stem lesion cerebral ischemia-reperfusion injury in rats_157

By Darlene Wood,2014-10-30 15:34
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Drinking game of the brain stem lesion cerebral ischemia-reperfusion injury in rats_157

Drinking game of the brain stem lesion cerebral ischemia-

    reperfusion injury in rats

     Abstract Objective To observe the brain stem to drink on focal cerebral ischemia-reperfusion brain injury in rats and to further explore its mechanism. Methods The middle cerebral artery embolization in rats produced focal cerebral ischemia-

    reperfusion injury model. The use of immunohistochemistry, respectively, TTC staining and Zea Longa 5-point scale scoring

    system to observe the brain stem in brain tissue of treated

    drinking Bcl 2 protein expression, infarct volume and neurological changes in behavior. Results Compared with model group, treatment group significantly reduced symptoms of neurological impairment (P <0.05); infarct volume reduced by

    about 40%; in each time point Bcl 2 expression in both the

    high and the model group (P <0.05). Conclusion brain stem drinking by Prostisol, Huoxue network of comprehensive control measures can not only increase apoptosis inhibitory gene Bcl

     2 expression, reduced infarct volume, but also can improve the symptoms of nerve injury, reducing structural damage brain tissue, lack of brain Blood-reperfusion injury caused

    significant protection.

     Key words Brain stem drink Bcl 2 protein in ischemia-

    reperfusion injury

     Experimental Study of the Protective Effects of Naogengyin on Cerebral Ischemia-reperfusion Injury in Rats

     Abstract: ObjectiveTo measure the protective effects of Naogengyin on cerebral ischemia reperfusion injury in rat

    and its mechanisms.MethodsThe model of focal reperfusion injury after cerebral ischemia in rats was made by middle cerebral artery occlusion. The effect of Naogengyin on

expression of Bcl 2 proin, cerebral infarct volume and

    behavioral change in rats brain tissue were observed by

    immunohistochemical method, TTC staining and Zea Longa method respectively.ResultsCompared with the control group, neurological grades in Naogengyin group were lower (P <0.05). The volume of cerebral infarction decreased 40% in treated

    groups. Bcl-2 expression of the treated groups increased more than that of the control groups in each hour point. There was great difference between the control groups and treated groups in expression of Bcl 2 protein (P <0.05 ).

    ConclusionNaogengyin has great cerebral protective effects

    against ischemia-reperfusion injury by increasing restrain mechanisms of the apoptosis of neuronal cell and expression of Bcl 2 protein, reducing the volume of cerebral infarction and damage to brain tissue.

     Key words: Naogengyin; Bcl 2protein; Ischemia


     Acute cerebral ischemia - reperfusion (IR) injury can

    cause severe neurological dysfunction, and even life-

    threatening, to find effective treatments and measures to promote functional recovery after stroke central nervous

    system has been the focus of the study of related disciplines . In recent years, Chinese medicine on the brain protective effect of more and more attention. Drink is a Professor Wang Youqi brain stem stroke prevention and treatment of clinical experience, common side, in the years of clinical use to obtain good effect. In this study, inhibition of apoptosis and reduced brain infarct volume point of view in the brain stem to drink in rats.

     1 Materials and methods

     1.1 Materials

     42 healthy Wistar rats, weighing 250 ~ 300 g. The brain stem to drink from Astragalus, habitat, 37, the Health Puhuang etc., these drugs are filtered decoction condensed into 100 ml (containing crude drug 1.2 g / ml). Bcl 2 monoclonal

    antibody, DAB chromogenic kit, HIGH SAB kit were purchased

    from Wuhan Boster Company.

     1.2 Methods

     1.2.1 Methods of modeling documents using the light of improved Koizumis method [1] Preparation of MCAO animal model. The external carotid artery ligation in rats, since the common carotid artery bifurcation to the carotid artery into plug-

    line (18.5 ? 0.5) mm, block the middle cerebral artery blood flow, resulting in focal cerebral ischemia. After the tail of the plug wire placed through the skin, ischemia and 2 h after

    the bolt of light pulling lines caused by blood reperfusion. Survival in rats observed after 6,12,24 h reperfusion in rats changes in behavior.

     Zea Longa behavioral ratings refer to the five-point

    scale score criteria: ? 0 points: normal, no symptoms of

    nerve injury; ? 1 pm: contralateral forepaw flexion or full flexion part; ? 2 points: the hemiplegic side turning in circles; ? 3 points: the hemiplegic side of dumping; ? 4

    points: You can not spontaneously walk, loss of consciousness.

    Sham-operated group, in addition to non-insertion lines, the

    remaining steps ibid.

     1.2.2 Grouping and administration will be making a successful model rats were randomly divided into sham operation group, model group and treatment group. In addition

    to sham-operated group, the other in each group is further divided into two groups after ischemia-reperfusion 6,12,24 h 3

    teams, a total of seven groups, the ultimate success of surgery combined 35 experiments in each group 5. Sham-

    operation group, model group and treatment group 1 week before surgery, respectively, by 1 ml/100 g / times, 2 times / d given continuously fed to drink salt water and the brain stem. Treatment group, 3 h before surgery and postoperative 3,6,12 h respectively of the gavage one time.

     1.2.3 Brain Bcl 2 protein in rats after ischemia-

    reperfusion, respectively, at different time points, excessive anesthesia, open chest, exposing the heart, with 4 ? heparin

    saline (2 × 105 u / L) 150 ml quick wash, followed by 0.1 mol

    / L phosphate buffer (pH7.4) prepared 4% paraformaldehyde perfusion-fixed 30 min, and then decapitated whole brain, after 4% paraformaldehyde 24 h, and then the gradient lines alcohol dehydration, xylene transparent, Baptist wax embedding. Levels in the SCN using microtome coronal slices, slice thickness 7 mm. Each interval of taking five sections,

immunohistochemical staining.

     Multimedia pathological image analysis system (CMIAS

    008 type, Beijing University of Aeronautics and Astronautics

    Research), the counts for each field of vision in the number of positive cells and negative cells, the final calculation of the percentage of positive cells, the percentage of positive cells = number of positive cells / (positive cell-negative

    cells) × 100%.

     1.2.4 Morphological observation of brain tissue to normal brain tissue by paraffin section method dehydrated, transparent, soaked wax, embedding, slicing done after HE staining, morphological changes observed in brain tissue.

     1.2.5 TTC (TTC) staining were decapitated, the brain

    removed from the extreme amount of 2.5 mm at an interval of 2.5 mm produced eight coronal slices. Will be placed in 2% slice chloride 2,3,5 triphenyl tetrazolium solution (TTC) in, 37 ? dark incubated 30 min, and then go to 0.1 mol / L

    PBS were prepared in 4% paraformaldehyde liquid fixed. Photography and application of computer image processing and analysis system to calculate an area of infarction for each slice, according to infarction size and slice distance of slices to calculate infarct volume.

     1.2.6 Statistical analysis All data were characterized by SPSS10.0 statistical software, which resulted in (? s), said

    analysis of variance for comparison between each group.

     2 Results

     2.1 The general observation model group and treatment

    group were on their own after a clear reduction in activity, with time to extend the autonomy of living increasing. The treatment group compared with the model group significantly speed up the recovery.

     2.2 The assessment of neurological function in rats sham-

    operated group had no abnormal behavior, behavioral score of 0 points; model rats appeared contralateral forelimb can not be extended to the paralyzed side of turning in circles or to the paralyzed side of dumping symptoms of nerve damage, behavioral

    score (1.8 ? 0.68). Treatment group, mild symptoms of nerve

    injury, only the contralateral forelimb can not be fully extended, the behavioral score (0.6 ? 0.47) min. The results

    in Table 1. Table 1 ischemia - reperfusion neurobehavioral

    score (abbreviated)

     2.3 The morphological changes of brain tissue of sham-

    operated group the number of nerve cells and more normal shape, contour clarity. Ischemia after 6 h, model group, part of the central area of ischemic neuronal lesion disappeared,

    there are more red cells and ghost cells and a small amount of reversible shape integrity of damaged cells; ischemia and 24 h, large areas of the brain model group appears tissue ischemia and necrosis, focal central area of neuronal cells in Tuo Shi, wider than 6 h increased proliferation of glial cells surrounding necrotic foci markedly. The treatment group at all time points to narrow the scope of both ischemic and necrotic area is also reduced infarct edge of the organizational structure is not completely destroyed, damaged neurons

    significantly reduced the number of nerve cells in the ischemic region increased significantly. Figure 1 ~ 6.

     2.4 Organization of infarct volume of brain tissue at TTC staining of necrotic tissue was pale color, no necrotic tissue

    red. Sham-operated group without brain infarction; model group ischemic brain tissue infarction phenomenon of brain infarct volume accounting for 36.10%; treatment group ischemic infarct volume compared with model group reduced 40% (P <0.05). The

    results in Table 2. Table 2 after ischemia-reperfusion in rat

    brain infarct volume comparison (omitted) reposted elsewhere in the paper for free download http://

     2.5 Bcl 2 protein expression in sham-operated group

    was no Bcl 2 protein expression. The remaining two groups were seen at each time point Bcl 2 protein expression,

    reperfusion 6 h, Bcl 2 positive cells in a small amount of expression, 12 h expression was significantly, 24 h expression of a further drop. Treatment group in each time point Bcl 2

    expression were higher than the model group, there are significant differences between the two groups (P <0.05), which increased to 12 h the most significant (see Table 3 and Figure 7 ~ 12). Table 3 Cerebral Ischemia two Bcl-2 positive

    cells in the percentage of comparison (omitted)

     3 Discussion

     Cerebrovascular disease in medicine are "stroke" "Pianku" "hemiplegia" "Stroke" and other areas. The etiology was mainly due to old and feeble, Qi and Yin Deficiency, blood stasis due

    to network resistance. This disease with Qi and Yin Deficiency-based, congestion resistance network for the standard, actual situation inclusions, qi deficiency blood stasis of disease throughout the entire process. Brain stem drank Prostisol, activating blood and network for the

    treatment of Dharma, mainly for the treatment of Qi, or Qi and Yin Deficiency diseases among the wind.

     Modern pharmacological studies have shown that [2]: Astragalus enhanced cell vitality and resistance, to enhance

    hypoxia tolerance in mice, as well as anti-oxidation, anti-

    aging effect; habitat with a vasodilator to improve blood rheology, increase cerebral blood flow, clear oxygen free radicals, anti-oxidation effect; 37 with anti-platelet

    aggregation, antithrombin and the promotion of fibrin

    degradation and anti-thrombosis and anti-oxidation, anti-free

    radical damage, promote the formation of collateral circulation infarct, microcirculation, membrane The role of the stability and other aspects.

     Cerebrovascular disease is harmful to human health, one of the three diseases. The damage mechanisms, including oxidative stress, excitatory oxy acid toxicity, inflammation, calcium overload and apoptosis and so on. In which apoptosis is delayed cerebral ischemia and reperfusion key to nerve cell


     Neuronal apoptosis involves the expression of a variety of factors out of control and regulation of anomalies in the nervous system, now known and apoptosis-related genes divided

    into two basic categories: one category is the promotion of

    apoptosis genes, such as bax, bcl xs, bad, bak, c fos, c

     jun, p53 and so on, and the other is the inhibition of apoptosis genes, such as Bc1 2, Bcl XL, BCl W, mcl

    1 [3]. One proto-oncogene Bcl 2 inhibit apoptosis features,

    is the first one has been identified as able to combat apoptosis gene, which specifically inhibit the cell apoptosis has attracted wide attention from scholars. Bcl 2

    inhibition of apoptosis through a variety of mechanisms: (1) a variety of anti-lipid peroxidation; (2) inhibition of

    mitochondrial release of pro-apoptotic protein; (3) antagonist pro-apoptotic genes; (4), inhibited the calcium overload; (5) inhibit the activation of cysteine protease-rich [4]. The

    experimental results showed that the brain stem drinking can

    significantly increase Bcl 2 protein expression, the

    treatment group at all time points Bc1 2 protein expression

    were higher than model group, which, in the 12 h, Bc1 2

    protein expression reached a peak. Description of Chinese

    medicine to drink can improve the brain stem induced by ischemia-reperfusion in neurons of Bcl 2 protein content

    was decreased, the promotion of neuronal Bcl 2 protein

    expression, thereby protecting neurons played a role in reducing cerebral ischemia-reperfusion of brain tissue damage, the brain stem to drink increases Bcl 2 protein expression

    in the mechanism has yet to be and further research.

     The study found a large number of experimental focal cerebral ischemia in Bcl 2 protein expression can reduce

    the infarct volume. Hata and other genes in the Bcl 2

    protein In addition to the drum 1 h focal cerebral ischemia in rats found that absence of Bcl 2 protein does not affect

    cerebral blood flow, but the infarct volume increased significantly. The experimental results showed that: the model group of brain infarct volume accounting for 36.1%, while the treatment group, only whole brain infarct volume 21.66% in treatment group compared with model group, infarct volume reduced by 40%, there are significant differences between the two groups (P < 0.05). This Pan Palace Shuqing [5] reported in the literature is consistent, and that structural damage in the brain tissue, the two groups there was a marked difference: Compared with model group, treatment group,

    infarct edge of the organizational structure has not been completely destroyed, the number of nerve cells in the ischemic region a marked increase in pyknosis reduction in the bubble-like changes in neurons decreased significantly. Note to drink with a significantly reduced brain stem infarct size and reduce ischemia-reperfusion induced brain injury in the organizational structure. Its mechanism may have the following aspects: ? by increasing the expression of Bcl 2 reduced

    infarct volume; ? inhibit Ca2 flow, reducing free radicals, reducing the death of nerve cells; ? increase in cerebral

    blood flow in ischemic areas; ? anti-platelet aggregation and


     In addition, the experimental results also found that the independent activities of treated rats compared with model group significantly speed up the recovery, and in the neurological function scores, the model group, apparent symptoms of neurological impairment, such as rats moved around to the paralyzed side, dumping can not even stand up or coma

    and other symptoms. The treatment of mild symptoms of nerve injury, only lateral forelimb can not be fully extended, a small number of circular motion to the paralyzed side, no dumping, coma and other symptoms. That the brain stem drinking

    can significantly improve the cerebral ischemia-reperfusion

    induced symptoms of nerve injury, which is the side Prostisol, promoting blood circulation meridians of the effectiveness of closely related.

     In short, through this experimental study, further

    confirmed the protection of the brain stem to drink the role of cerebral ischemia-reperfusion injury, which can in many

    ways, multi-angle, multi-target to play against ischemia-

    reperfusion injury of brain cells, which expand the application for clinical provide a reliable theoretical and experimental basis.


     [1] Nagasawa H, Kogure K. Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion [J]. Stroke, 1989,20:1037.

     [2] WANG Ben-Xiang. Modern TCM Pharmacology [M]. Tianjin: Tianjin Science and Technology Press, 1999:1175.

     [3] Macaya A, Apoposis in the nervou system [J]. RevNeurol, 1996,24 (135): 1356.

     [4] Ma Zhong, XUE Rong-liang. Bcl 2-related proteins

and cell apoptosis and cerebral ischemia-reperfusion injury

    [J]. "Foreign Medical" anesthesia and recovery volumes, 2001,22 (6): 362.

     [5] Pan Dian-qing, LI Zhi-mei. Neuroprotective agents

    cocktail rat infarct volume after focal cerebral ischemia and

    neuronal apoptosis and protein expression [J]. Chinese Journal of Neurology, 2004,35 (2) : 75. reposted elsewhere in the paper for free download http://

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