The Role Of Ceftriaxone In Early Brain Injury After Subarachnoid Hemorrhage In Rats

BackgroundAlthough aneurysmal subarachnoid hemorrhage (aSAH) represents only 5% of all strokes, it causes significant burden to society, given its high mortality and morbidity, lasting poor clinical outcome and the young age of its onset. Delayed cerebral vasospasm, first reported by Ecker et al. in 1951, is considered to be the main cause of the poor outcome of the aSAH. Although considerable studies targeting vasospasm have been done to reveal the mechanisms underlying aSAH, no definitively effective method is established to improve the outcome of aSAH. In recent years, emerging evidence indicated that onset of cerebral vasospasm possibly dissociate with the poor neurological outcome of aSAH. It is proved by the poor relationship between vessel constriction and cerebral infarction and the disproportionate incidences of angiographic vasospasm and delayed cerebral ischemia (DCI). Furthermore, an ET-1A antagonist, clazosentan confirmed to alleviate cerebral vasospasm post-aSAH failed to improve the prognosis in aSAH patients in two randomized, placebo-controlled, double-blind, phase III trials (CON-SCIOUS-2 and CONSCIOUS-3), corroborating the concept.Recently, several new mechanisms of aSAH have been revealed, among which is early brain injury (EBI). EBI, occurs within 72h after SAH, appears to be a principal factor accountable for poor outcome of SAH patients, involving complex mechanisms including increased permeability of BBB, cell apoptotic activities and excitotoxicity of extracellularly accumulated glutamate.Glutamate, known as the principal excitatory neurotransmitter, is neurotoxic even at micromolar concentrations. Local accumulation of glutamate from plasma and red blood cells extravasated to the brain contributed to brain insults after SAH. Excitatory amino acid transporter2 (EAAT2), also known as Glutamate transporter 1 (GLT1) in rodents, is the dominant EAAT which is critical in the clearance of synaptic glutamate by transporting it back to intracellular space, but was reported to decrease in neurological disorders, including stroke, amyotrophic lateral sclerosis (ALS) and epilepsy. Thus, repressing the expression of EAAT2 to prevent glutamate neurotoxicity seems promising in SAH treatment. β-lactam antibiotics were stimulators in expression of EAAT2 as previously described. Ceftriaxone, the most efficient P-lactam antibiotic in crossing blood-brain barrier, was reported to exert neuroprotection by increasing EAAT2 expression in animal models of ALS, stroke and traumatic brain injury. All these studies implied that ceftriaxone may provide potent neuroprotection in SAH.In our study, we tested the neuroprotective effect of ceftriaxone on the EBI following 24h of SAH by investigating whether ceftriaxone reduces mortality, represses EAAT2 expression, improves neurological outcomes, attenuates apoptosis and stabilizes BBB.Experimental Methods1. Endovascular perforation was used to perform SAH model. Adult male Sprague-Dawley rats were randomly divided into Sham+Vehicle group, SAH+Vehicle group and SAH+CTX group.24h after SAH was chosen as the time point of all the investigated endpoints including SAH score, mortality, neurological score and brain water content to assess early brain injury of the SAH rats.2. Ceftriaxone was injected intraperitoneally into adult male rats consecutively for 5 days before the performance of endovascular perforation. Adult Sprague Dawley rats were randomly assigned into Sham+Vehicle group, SAH+Vehicle group and SAH+CTX group.24h after SAH, western blotting was performed to assay the expression of EAAT2, MMP9, p53, Bax, Bcl-2, occluding, claudin5 and IL-1β.Brain edema was evaluated quantitatively by calculating average water content of brain samples and extravasation of Evans blue in each group.3. Adult Sprague Dawley rats were randomly assigned into Sham+Vehicle group, SAH+Vehicle group and SAH+CTX group.24h after SAH,apoptosis was assayed by western blotting of apoptosis-related protein caspase-3 and TUNEL/DAPI staining. The activation of microglia was evaluated by Iba-/DAPI immunohistological staining.Result1. Pretreatment of ceftriaxone reduced the mortality of SAH rats. In comparison with the SAH+vehicle rats, SAH+ceftriaxone rats showed a significantly less severe neurological deficit in response to the surgery. Ceftriaxone also statistically alleviated brain edema demonstrating a BBB-protective effect. It is noteworthy that there was no difference in the grade of SAH between groups.2. EAAT2,occluding,claudin5 and Bcl-2 expressions in the ceftriaxone-treated group increased significantly compared with those in SAH+vehicle group, attenuating the neurotoxicity of glutamate and BBB disruption after SAH. Ceftriaxone statistically reduced cerebral expressions of Bax, p53, caspase-3, MMP-9, IL-1β and extravasation of Evans blue.3. Pretreatment of ceftriaxone decreased TUNEL-positive cells 24h after SAH. Also,. The immune-histological staining result revealed the ceftriaxone significantly suppressed the activation of microglia.Conclusion1. Ceftriaxone pretreatment provided neuroprotection in EBI, improving neurological outcome, reducing mortality, attenuating brain edema in rats 24h after SAH.2. Ceftriaxone pretreatment inhibited expression/activation of MMP-9, reversed the reduction of EAAT2, suppressing microglial activation, blood brain barrier disruption and neurotoxicity in rats 24h after SAH.3. Ceftriaxone pretreatment suppressed cell death in EBI by inhibiting mitochondrial apoptosis.