The Effect And Mechanism Of Ultra Low Molecular Weight Heparin On Cerebral Ischemia/Reperfusion Injury In Rats

Objective: Cerebrovascular diseases have the characteristics of high morbidity, high rate of mutilation and high mortality. With the aggravation of population aging, ischemic cerebrovascular disease (ICVD) is becoming one of the main diseases which has a serious impact on human health and the lives. It has caused global attention. The patho-physiological mechanisms of cerebrovascular diseases haven't been fully clarified till now and there is currently no very effective treatment. This study is to explore the antagonistic effects of ULMWH on focal cerebral ischemia-reperfusion injury using generally acknowledged model of cerebral ischemia, with the neurological score, percent of edema and infarct volume and pathological morphological changes of brain as the main assessment index. The possible mechanism of ULMWH was elucidated by investigating energy metabolism, oxidative stress, calcium overload, excitatory amino acid, inflammatory reaction, apoptosis and relative gene expression from rat brain cells in the focal cerebral ischemia-reperfusion injury. This study might have an important implication in ULMWH growing into a newly drug for the future treatment of the ischemic cerebrovascular diseases.Methods: Typical models of brain ischemia-reperfusion injury were established by middle cerebral artery occlusion (MCAO) in rats. All animals were randomly divided into five groups: model group, ULMWH-treated group (1 and 0.5mg kg^-1, i. v.), LMWH-treated group (1mg kg^-1, i. v.) and sham group. Drug was dissolved in saline, and administrated twice 10 min after the MCA occlusion and reperfusion. Saline was given to model and sham groups in the same administration. The present article is composed of four parts as follows.1. Antagonistic effects of ULMWH on focal cerebral ischemia-reperfusion injury(1) Neurological and body weight examination: Neurological evaluation was performed 24 h after induction of reperfusion and scored on a 5-point scale. After the examination, the rats were weighed and the percentage of body weight decreasing was calculated.(2) Measurement of edema and infarction volume: 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining method was used to assess the percentage of brain edema and infarct volume.(3) Histopathological examination: After 24 h of reperfusion, rats were perfused with physiological saline and then with 4% paraformaldehyde (PFA) solution in 0.1 M phosphate-buffered saline (PBS). Brains were removed, post-fixed in 4% PFA overnight. The specimens were dehydrated with gradient alcohol, made transparent by dimethylbenzene, and embedded in paraffin routinely to prepare serial sections of 4μm. Sections were stained by hematoxylin and eosin. Pathologic histological changes were observed by optical microscope.2. Antagonistic Effects of ULMWH on the injury of nerve cells(1) Cerebral energy metabolism: the ischemic cortex was homogenized in saline. Colorimetric method was used to determine the changes of lactic acid (LD) level and ATPase activity.(2) Cerebral lipid peroxidation level and antioxidase activity: Colorimetric method was used to determine the changes of malondialdehyde (MDA) level, superoxide dismutase (SOD) and Glutathione peroxidase (GSH-PX) activities. (3) Measurement of glutamic acid content: Colorimetric method was used to determine the change of glutamic acid content.(4) Measurement of the intracellular Ca²⁺ concentration ([Ca²⁺]i): Double wavelength fluorospectrophotometry was used to assay the intracellular Ca²⁺ concentration by incubating the ischemic hippocampal cells with the fluorescent Ca²⁺ indicator Fura-2/AM.3. The effects of ULMWH on inflammatory reaction(1) Observation of microglia: Microglia proliferation and neuronophagia were observed by optical microscope.(2) Examination of Nuclear factor kB (NF-kB) protein: The expression of NF-κB protein in hippocampus was assessed by immunohistochemistry. The deparaffinized sections (2μm) were immunostained with antibodies to NF-kB (mouse monoclonal) and horseradish peroxidase (HRP) labeled anti-mouse secondary antibody. Immunoreactivities were demonstrated by DAB method.(3) Measurement of ICAM-1 mRNA expression: The ischemic hippocampus tissue was isolated from the brain. Total RNA was extracted using trizol reagent. 2μg of total RNA was used for detecting the mRNA level.β-actin was used as reference.4. The effects of ULMWH on the apoptosis of nerve cells(1) Quantification of apoptosis in hippocampus: hippocampus of ischemic hemisphere is made into cell suspension. After fixation, cells were stained with DNA specific fluorochrome propidium iodide (PI). The PI fluorescence of nuclei was measured using flow cytometry.(2) Measurement of Caspase-3 mRNA expression: The ischemic hippocampus of brain tissue was isolated. Total RNA was extracted using trizol reagent. 2μg of total RNA was used for detecting the mRNA level.β-actin was used as control.Results:1. Antagonistic effects of ULMWH on focal cerebral ischemia-reperfusion injury (1) Neurological and body weight examination: neurological deficit scores of model group were significantly higher than those of the sham group. Treatment with ULMWH at dose of 1mg kg^-1 significantly decreased neurological deficit scores compared with the model group. Moreover, it showed a much better potency compared with the treatment with LMWH at the same dose. The model group showed a significant decrease in body weight compared with the sham group. Treatment with ULMWH at the two doses, but not with LMWH, significantly inhibited the decrease of body weight.(2) Measurement of edema and infarction volume: The brain of sham group showed rose red, while the model group showed great infarct area in cortex, striatum and hippocampus. The two hemispheres were not symmetric and the injured side was obviously swollen. Treatment with ULMWH at both doses extremely significantly reduced the infarct volume. At the same dose (1mg kg^-1), it showed a much better potency compared with LMWH treatment. As for depressing edema, ULMWH also exhibited significant effect.(3) Histopathological examination: The sham group revealed healthy neuronal cells. The structure of neuron in model group was wrinkled and obviously edema, the cytoplasm presented acidophilic staining, the nucleus dyed deeply or disappeared. The arrangement of pyramidal neurons in hippocampal CA1 region was not in order and the amount was decreased. ULMWH can alleviate neuronal damage obviously. The shape of neurons was almost normal and the pathological changes of pyramidal neurons in hippocampal CA1 region significantly relieved.2. Antagonistic Effect of ULMWH on the injury of nerve cells(1) Energy metabolism: LD level was much higher and ATPase activity was much lower in the model group than those in the sham group. The treatment with ULMWH or LMWH decreased LD level and increased ATPase activity.(2) Lipid peroxidation level and antioxidase activity: SOD and GSH-PX activities were much lower and MDA level was much higher in the model group than those in the sham group. The treatment with 1mg kg^-1 ULMWH increased SOD and GSH-PX activities compared with the model group. At the same time, the level of MDA was significantly reduced.(3) Measurement of glutamic acid content: Glutamic acid content of model group was significantly higher than that of the sham group. The treatment with ULMWH or LMWH decreased glutamic acid content.(4) Measurement of the intracellular Ca²⁺ concentration: Compared with the sham group, the intracellular Ca²⁺ concentration of model group increased significantly. Compared with model group, the treatment with ULMWH or LMWH could decrease [Ca²⁺]i and ULMWH showed a better potency compared with LMWH at the same dose of 1mg kg^-1.3. The effects of ULMWH on inflammatory reaction(1) Observation of microglia: There is few Microglia in the sham group. Proliferation and neuronophagia of Microglia were observed in model group. ULMWH can inhibit Microglia proliferation and neuronophagia.(2) Examination of NF-κB protein: In the sham rats, only few neurons expressed NF-κB, while the model group showed obvious positive cells in cortex and dentate gyrus of hippocampus. Positive neurons showed brown staining in nucleus. The treatment with ULMWH or LMWH decreased the expression of NF-κB and the positive neurons were stained in cytoplasm. ULMWH showed a better potency compared with LMWH at the same dose of 1mg kg^-1.(3) Measurement of ICAM-1 mRNA expression: The expression of ICAM-1 mRNA was much higher in the model group than that in the sham group. The treatment with ULMWH or LMWH decreased the expression significantly. ULMWH showed a better potency compared with LMWH at the same dose of 1mg kg^-1.4. The effects of ULMWH on the apoptosis of nerve cells(1) Quantification of apoptosis in hippocampus: The apoptosis percentage was much higher in the model group than that in the sham group. The treatment with ULMWH or LMWH decreased the percentage significantly.(2) Measurement of Caspase-3 mRNA expression: The expression of Caspase-3 mRNA was much higher in the model group than that in the sham group. The treatment with ULMWH decreased the expression significantly. ULMWH showed a better potency compared with LMWH both at the dose of 1mg kg^-1.Conclusions:1.ULMWH exhibited significant antagonistic effects on focal cerebral ischemia-reperfusion injury by decreasing neurological deficit scores, increasing the body weight and reducing the edema and infarct volume.2.ULMWH can improve cerebral energy metabolism, decreasing intracellular Ca²⁺ concentration, inhibit the oxidative stress and Glu releasing,3.ULMWH can inhibit Microglia proliferation and neuronophagia in cortex. ULMWH can also decrease the protein expression of NF-κB and inhibit its activation. This might lead to the decreasing of the expression of ICAM-1 mRNA.4.ULMWH can inhibit apoptosis which might be partly mediated by decreasing the expression of Caspase-3 mRNA.