| Background:Intracerebral hemorrhage(ICH) is a neurological disease with urgent onset, rapid progress, life-threat and high recurrence rate. Patients with ICH often suffered severly neurologic sequelae, such as hemiplegia, aphasia and so on. ICH not only brings great suffering to patients, but also leaves the family and society with the financial burden. So It is extremely vital significance to prevent ICH, particularly to recover the central nervous system functions and reduce sequelae caused by never damage. Nevertheless, there has been no ideal treatment for ICH. The acute phase is key to dealing with ICH. Conventional therapies focus on reducing the intracranial pressure, maintenance of vital signs, symptomatic treatment, applying hemostatic drugs and surgery.Edaravone is a newly developed free radical scavenger, which can inhibit brain edema, improve neurological function, reduce the permeability of the blood brain barrier and relieve associated neurological symptoms. It has been employed in the treatment of ischemic stroke in clinic and works well. Edaravone can improve the function and local structure of the ischemic brain tissue. Experimental cerebral ischemia in rats indicated that Edaravone could reduce NO production induced by ischemia and reperfusion, and improved significantly neurological function and decreased ischemic area. Both cerebral ischemia and ICH can both reduce regional brain tissue oxygen supply and result in nerve tissue damage. Intracerebral hemorrhage(ICH) has the similar pathological process as ischemic stroke but not identical. The reason for the relationship is not yet known. They both produce large amounts of free radicals, cause inflammation factors storm, activate macrophages and microglial cells and promote neuronal apoptosis. In the rat and mouse model of cerebral hemorrhage, edaravone can reduce neuronal apoptosis induced by cerebral hemorrhage to improve brain metabolism, reduce nerve damage and brain edema caused by cerebral hemorrhage. Up to now, clinical research and mechanism exploration of ICH are far less than ischemic infarction reported. The molecular mechanisms of regulation and anti-inflammatory network are not yet clear. Therefore, a further study is needed to clarify the clinical effect of edaravone on ICH and the associated molecular mechanism.There are many ways to adminstrate edaravone. Different routes of administration affect theraputic effect. How to quickly increase the drug concentration at the target site is crucial to improve the efficacy of edaravone. Intravenous injection or infusion administration is mainly used and arterial administration is rarely reported in clinic. Besides, intraperitoneal administration is mainly used in animal model studies. It is not reported that intracranial topical administration is used in clinic. Intraperitoneal administration will lead to drug consumption for mesenteric absorption and transfer into the blood. Drugs can be directly into the circulating system by intravenous infusion and arterial injection without filtering through the gut wall and liver. Compared with the intraperitoneal route of administration, it can obtain faster and higher plasma concentration. However, drugs must be distributed in the circulating system before ultimately reaching the target site, which reduced the concentration of edaravone and increased the probability of side effects. Besides, intravenous injection needs to insert the needle into the vein which is hard to find in some patients, especially in obese patients. For that matter, we need to find a more effective route of administration. Stereotactic administration is presently considered to be a more effective administration, which is particularly suitable for patients with a little intracranial bleeding where minimally invasive stereotactic surgery or conservative treatment required for topical treatment. It avoids the long time and distance blood delivery and is not affected by the blood-brain barrier. But is it safe to use edaravone by stereotactic injection? Is stereotactic administration better than other routes of administration? Which mechanism is activated to improve neurological function through stereotactic administration? These issues are discussed in this article.Objectives:(1) Observe the neuroprotection function of edaravone administrated by stereotactic injection in rats with ICH.(2) Observe the mechanism of edaravone stereotactic injection regulating inflammatory responses and apoptosis.(3) Compare the efficacy of edaravone between different administration routes.Materials and methods:Part I: Neuroprotective effects of edaravone stereotactic administration in rats with ICHModeling and groups:In this study, 201 homologous adult SD rats(male) weighted 250-280 g, were randomly devided into 4 groups: sham operation group, saline control group, intraperitoneal injection of edaravone treatment group and stereotactic injection of edaravone treatment group. ICH was induced by stereotactically infusing collagenase VII into the caudate nucleus. Saline control group: To inject 10 μL saline into hemorrhage cavity at 24 h and 72 h after ICH.Intraperitoneal injection of edaravone treatment group: To inject edaravone of 3 mg/kg into peritoneal cavity at 1 h, 24 h, 48 h and 72 h post-ICH. Stereotactic injection of edaravone treatment group: To inject 10 μL edaravone of 1.5 mg/ml into hemorrhage cavity 24 h and 72 h after ICH.Index evaluation:(1) Behavior evaluation: Neurobehavioral scores(mNSS) were recorded at different time points in the four groups;(2) Lesion volume of brain tissue: To evaluate the lesion degree by the hematoma volume and ipsilateral ventricle volume rebuilded throught MRI three-dimensional reconstruction. And histopathology was accessed by the HE staining;(3)Regeneration of nerve fibers: Fast Blue staining and NF200 immunofluorescence staining show remyelination and axonal regeneration;(4) Glial scar formation: To observe the glial scar formation by GFAP immunofluorescence staining.Statistical analysis:All values are expressed as mean ± standard deviation(SD). Differences between groups were tested for statistical significance using one-way factorial analysis of variance(ANOVA). After modeling, the data at each time point for each group of rats were tested using general linear model(GLM) for repeated measures. Comparison between different groups using one-way ANOVA analysis at every single time point and the Bonferroni correction was used to counteract the problem of multiple comparisons. A P< 0.05 was considered statistically significant(two-sided test, α = 0.05). All statistics are using the SPSS 21.0(SPSS Inc., Chicago, IL) and plots were drawn by prism 6.0.Part II: Regulation of edaravone stereotactic administration on inflammatory edema responses and apoptosis in rats with ICHModeling and groups:The same as the part I.Index evaluation:(1) Inflammatory edema reactions: 1) Cerebral edema assessment: To detect the ratio of brain tissue wet weight to dry weight; 2) Assessment of blood-brain barrier damage: Evans Blue staining and transmission electron microscopy; 3) Microglia polarization assay: microglia markers(iNOS/CD11 b, Arginase-1/CD11b) double staining, the protein level quantified by Western blotting accessed differentiation of microglia, to M1 or M2; 4) Expression of inflammatory factors: To test inflammation associated polarization factor(TNF-α, IL-1β, IL-4, IL-10) in serum and brain microglia using ELISA semi-quantitative detection method.(2) Apoptosis study: 1) To detect MDA content and assay apoptosis of nerve cells by TUNEL; 2) To evaluate levels of apoptosis-related protein, Caspase-3 and Bcl-2,by immunofluorescence staining.Statistical analysis:The same as the part I.Results:1. Behavior score was significantly lower in edaravone treatment group(edaravone stereotactic administration group and edaravone intraperitoneal administration group) than stereotactic saline control group 1.5 days after ICH(P< 0.05). And it is lower in stereotactic administration than the intraperitoneal injection 7 days post-ICH(P< 0.05).2. MRI showed that brain hematoma volume was significantly less in stereotactic than intraperitoneal injection and the saline control group 28 day post-ICH(P < 0.05). At day 7 and 14 after surgery, HE staining showed hemorrhage volume in stereotactic treatment group is less than in the saline controland intraperitoneal treatment group.3. Myelin deficiency degree, which is assayed by Fast Blue and immunofluorescence, is dramatically lower in the stereotactic injection group than in intraperitoneal injection and saline groups at day 7 and 14(P< 0.05). Meanwhile, NF200 level in brain tissue increases markedly(P< 0.05) and GFAP decreases significantly(P< 0.05).4. BBB permeability(Evans Blue dye) and tissue water content: They were significantly lighter than the saline control group at 5 days(P< 0.05). And edaravone stereotactic administration was lighter compared with intraperitoneal administration(P< 0.05). Astrocyte foot processes emeda observed by transmission electron microscope decreased significantly in edaravone stereotactic administration group than in other groups at day 14 and 28 post-ICH.5. Microglia marker staining showed Arginase-1(anti-inflammation type M2) high expression and iNOS markers(proinflammatory type M1) low expression. Western blot experiments showed that on 7 and 14 days after injection compared with the control group, Arginase-1 level was significantly higher and iNOS significantly lower(P< 0.05) in edaravone treatment group.6. Compared with edaravone treatment group(edaravone stereotactic administration group and intraperitoneal administration group), TNF-α and IL-1β expression was significantly lower than the control group(P< 0.05), but IL-4 and IL-10 expression was significantly higher than the saline control group(P< 0.05). Compared with intraperitoneal administration, TNF-α and IL-1β expression was significantly lower in stereotactic administration group, but IL-4 and IL-10 expression was significantly increased(P< 0.05).7. At day7 after ICH, down-regulation of Caspase-3 and upregulation of Bcl-2 proteins were significant in edaravone treating groups. Apoptosis cells decreased significantly compared with the saline control group(P< 0.05).8. Rats lived throught edaravone stereotactic injection process. There were not conclusions, infection and complications.Conclusions:This study demonstrates that edaravone by stereotactic injection can achieve better curative effect than by intraperitoneal injection in rats with ICH. Stereotactic administration can improve markedly ICH-induced neurological deficits in rats. The mechanism is mainly involved in the neuroprotective function, anti-inflammatory and anti-apoptosis effects. This study enriched the theoretical basis of edaravone treating intracerebral hemorrhage and provided a new administration rout for edaravone therapy in clinic. |
PDF Full Text Request