| Stroke is a lethal cerebral vascular disease caused by cerebrovascular suddenrupture or due to blockages in blood vessels which leads to cerebral ischemiaand hypoxia. The incidence of ischemic stroke patients is up to62.4percent,which makes ischemic stroke becomes one of the major killers to our health.Every year, almost1.5million patients died of ischemic stroke and an average ofevery15seconds there is a new case emerges. Nowadays, the most usefultreatment of ischemic stroke mainly rely on early detection and thrombolytictherapy. However, due to its limited time window, the majority of patients beforecarrying out effective treatment has lost the best timing of treatment, eventuallyleading to life-long disability or a series of complications which caused a hugeburden to our families and society. Focus on the mechanism of ischemic strokeand injury, find out reasonable and effectively ways to prevent and treat them, isa key method to control the incidence of ischemic stroke.Currently，the researches on the mechanism underlying ischemic braininjury are mainly concentrated on the neural excitotoxicity, oxidative stress,apoptosis, and inflammation. The inflammatory response is one of the maincauses associated with ischemic brain damage and cerebral reperfusion injury.As large number of studies have shown that after focal brain injury andreperfusion injury, the cascade of inflammatory response occurs, and the bloodcells, astrocytes and microglia are activated, as a consequence, a large numberof inflammatory factors such as IL-1, IL-1β IL-6and TNF-are released andinfiltrated into the brain tissue. Moreover, the inflammatory factors gather around the damaged brain blood vessels and further aggravate cerebral ischemia.How to find effective method to inhibit the activation of inflammatory cells,inhibit the release of inflammatory factors, block inflammatory damage iscrucial to treatment of the ischemic brain injury.Beta-lactam antibiotics are a clinically used broad-spectrum antibiotic, itsantibacterial effect and pharmacological mechanism has been clinically proven.New research shows that β-lactam antibiotics such as ceftriaxone (CTX) notonly has an antibacterial effect but also play a neuroprotective effect againstexcitatory glutamate damage to nervous system diseases such as amyotrophiclateral sclerosis (ALS), stroke, brain tumors, epilepsy and other diseases. Themechanism may due to increasing brain glutamate transporter-1(GLT-1).Meanwhile, the researchers observed inflammation also have some relief amongthe animal model or the patients, with varying degrees of recovery. Suchconclusion strongly suggests that β-lactam antibiotics may play a role inreducing the inflammatory injury.Therefore, we first proposed the following hypotheses:(1) After cerebralischemia and reperfusion injury, β-lactam antibiotic ceftriaxone can partiallyinhibit the activation of inflammatory cells such as astrocytes and microglia andplay a neuroprotective effect on the injuery.(2) In addition to the increase of theGLT-1, β-lactam antibiotics ceftriaxone can inhibit the releasing of criticalreperfusion injury inflammatory factor IL-1β, to reduce brain tissue damage.Based on the above hypotheses, this study aims to further explore theneuroprotective mechanisms of β-lactam antibiotics ceftriaxone.Objective：Based on the middle cerebral artery occlusion (MCAO) in rat, wewant to explore the neuroprotective mechanism of β-lactam antibioticsceftriaxone, confirming the relationship between and inflammatory cell activation and inflammatory cytokine release, in order to further reveal thepathogenesis of ischemic brain injury and provide new ideas of prevention andtreatment measures.Methods:72Sprague Dawley (SD) rats were randomly divided into modelgroup (n=60) and sham operation group (Sham, n=12). The model group wasdivided into a control group (Control, n=30) and ceftriaxone pretreated group(n=30). Ceftriaxone was injected intraperitoneally. After traditional middlecerebral artery occlusion (MCAO), we observe the neurological deficit score(NDS) and to calculate the infarct volume to determine the degree of the braintissue damage; Immunofluorescence staining after MCAO3h and24h was usedto observe astrocytes, microglia, neurons and the expression of interleukin1-β(IL-1β). Cells positive count was used to judge the activation of glial cells.Double labeling was used to judge the secretion of IL-1β. By ELISA andWestern Blot we detect after3h and24h the secretion changes of IL-1β in braintissue.Results: After MCAO, due to ischemia and reperfusion injury, the neurologicaldeficit score decreased, we can see obvious difference between the sham group,control group and the ceftriaxone pretreatment group (p <0.05), respectively,17.700±0.153,8.400±.349,12.000±0.447. With severe postoperative brainedema and infarct size increases, the main infarction area concentrated on thestriatum and some cortex after24h. Striatal infarction core area almost nodifference. The control group and the ceftriaxone pretreatment group infarctsize respectively (5.343±.211)%and (4.662±1.259)%, while the corticalinfarct size is in in difference (p <0.05), control group and ceftriaxonepretreatment group (32.748±1.802)%and (18.92±1.977)%.3h after MCAO,astrocyte activation is increasing and double stained with IL-1β, sham operation group, the control group and ceftriaxone pretreatment group astrocyte countswere44.020±3.882,167.300The±9.062and83.83±7.578. Western blotdetection of cortical IL-1β expression levels were1.020±0.041,3.017±0.076,3.108±0.107. After24hours of MCAO, activated microglia significantlyincreased, and double stained with IL-1β, sham operation group, the controlgroup and the ceftriaxone pretreatment group microglia counts were42.920±5.791,138.700±15.700and70.670±10.460. Western Blot cortical IL-1βexpression levels were1.020±0.041,3.178±.144and2.195±0.082. ELISA fordetection of cortical IL-1β expression levels were13.340±2.893,69.420±21.630and32.649±14.040.Conclusion: After ischemic reperfusion injury, brain tissue edema, the neuronsgradually necrosis, inflammatory cell infiltration. In the early stage of the injury,lots of astrocyte activate and release of inflammatory cytokines IL-1β whichmediated injury, while IL-1β expression levels rapidly increase. The effect ofCeftriaxone is not that obvious.24hours later, microglial activate andparticipate in the inflammatory injury, activation and release of IL-1β, in thisstage Ceftriaxone can be partially reversed by reducing microglia activation,reduce the secretion of IL-1β, and thus reduce the brain tissue damage. Byintraperitoneal injection of Ceftriaxone in advance, CTX can play an importantrole in the brain ischemia and reperfusion injury by controlling one of the majoraspects inflammatory response, partial reversal of infarct volume and improveneurological defect score, play a neuroprotective role in ischemic reperfusion. |
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