Cysteinyl Leukotrienes Induced Brain Injury And The Possible Mechanisms

Pathogenetic factors in the second phase of various brain injuries are believed to lead to brain damage. These factors include brain ischemia, excitotoxicity, production of free radicals, inflammation and apoptosis. Furthermore, the inflammatory reactions during brain injuries promote the development of brain damage. Cysteinyl leukotrienes (CysLTs, including LTC4, LTD4 and LTE4) are 5-lipoxygenase (5-LOX) metabolites of arachidonic acid. CysLTs are the potent inflammatory mediators involved in the central nervous diseases including cerebral ischemia, brain trauma, brain tumors, epilepsy, in addition to peripheral inflammatory diseases, such as asthma and allergic rhinitis. It has been reported that the production of CysLTs in the brain is increased after cerebral ischemia. 5-LOX inhibitors reduce the release of CysLTs and ameliorate cerebral ischemic injuries. These findings indicate that CysLTs are involved in injuries after cerebral ischemia. However, it is unknown whether CysLTs posses direct effects on brain damage, and which of the CysLT receptor subtypes are involved in the CysLTs caused brain damage.Brain injury is a highly complex process. As a main pathological change, brain edema occurs following a variety of injuries including brain trauma, stroke andtumors. The two main types of brain edema are vasogenic and cytotoxic edema. Vasogenic edema occurs when blood-brain barrier (BBB) is disrupted, which permits plasma fluid into the brain extravascular space; while cytotoxic edema occurs when water flows from the vascular compartment through intact BBB and astrocytic foot processes, and accumulates primarily in astrocytes. Recently, aquaporins (AQPs) are found to be the molecules responsible for the formation and absorption of excess fluid in tissue edema, including brain edema.AQPs are a family of transmembrane proteins that selectively allow the passage of water through the plasma membrane. Aquaporin-4 (AQP4) is the major water channel in the brain, and involved in brain edema formation after various brain injuries. In APQ4-deficient mice, the distinct roles of AQP4 have been proven in cytotoxic and vasogenic brain edema. It is known that CysLTs/CysLT receptor mediates neuronal damage, and is involved in BBB dysfunction and brain edema. Therefore, it is important to clarify whether CysLTs-induced post-ischemic brain edema is mediated via enhancing AQP4 expression.To clarify the role of CysLTs in the brain injury, we investigated the effects of CysLTs on BBB disruption, brain edema, neuron injury and cortical AQP4 expression 24 h after cortical LTC4 or LTD4 microinjection. We also observed the effect of exogenous LTD4 on the expression of AQP4 in both mice cortex and rat primary astrocyte cultures, and clarified the subtype of CysLT receptors involved in the up-regulation of AQP4 expression.Part IIntracortical microinjection of CysLTs induces cerebral damage in miceThe production of CysLTs in the brain is increased after cerebral ischemia. 5-LOX inhibitors reduce the production of CysLTs and ameliorate cerebral ischemic injuries. In addition, we have also found that CysLT₁ receptor antagonists (pranlukastand montelukast) protect against cerebral ischemic injury in rats and mice. These findings indicate that CysLTs are involved in injuries after cerebral ischemia. To further clarify whether CysLTs directly induces brain injury and which subtype of CysLT receptors is involved in this injury, we determined the effects of exogenous LTC4 and LTD4, potent agonists of both CysLT₁ and CysLT₂ receptors, in mouse brain. The cerebral injuries and cortical AQP4 expression were induced by cortical LTC4 or LTD4 microinjection in mice. Infarct areas, brain edema, survival neuron density, degenerated neuron density and IgG extravasation (BBB dysfunction) were determined 24 h after the microinjection. Moreover, the dose- and time-dependencies of the effects of CysLTs were also assessed. We found that LTC4 and LTD4 (> 1 ng in 0.5 μl PBS) caused cerebral injury in dose- and time-dependent manners as determined by TTC staining. LTD₄ 1 ng induced cerebral edema, increased the degenerated neuron density and IgG extravasation, and enhanced AQP4 expression. These responses were attenuated by 0.1 mg/kg pranlukast, a CysLT₁ receptor antagonist, injected 30 min before and after cortical microinjection. However, pranlukast did not significantly affect both LTL₄-induced brain edema and up-regulation of AQP4. Thus, we conclude that CysLTs cause brain injury through CysLT₁ receptor-mediated BBB dysfunction and the resultant vasogenic brain edema. Furthermore, CysLTs may promote the formation of cytotoxic brain edema via cortical AQP4 up-regulation that may be mediated via other mechanisms than CysLT₁ receptor.Part IILeukotriene D₄ enhances CysLT₂ receptor-mediatedaquaporin 4 expression in rat primary astrocyte culturesAQPs are a family of transmembrane proteins that selectively allow the passage of water through the plasma membrane. Aquaporin-4 (AQP4) is the major waterchannel in the brain. APQ4 is expressed at fluid-tissue barriers (blood-brain and rain-cerebrospinal fluid barriers) throughout the brain and spinal cord, suggesting its role in water transport under normal and pathological conditions. In APQ4-deficient mice, the distinct roles of AQP4 have been proven in the two main types of brain edema, cytotoxic and vasogenic edema. AQP4 deficiency reduces the cytotoxic edema induced by focal cerebral ischemia, water intoxication and bacterial meningitis, but worsens the vasogenic (fluid leak) edema induced by brain tumor, brain abscess, cortical freeze-injury and hydrocephalus. In the Part I of the present study, LTD4, a potent agonist of both CysLT₁ and CysLT₂ receptors, was found to up-regulate the expression of cortical AQP4, which was not inhibited by pranlukast, a CysLT₁ receptor antagonist. To further confirm the effects of CysLTs on the regulation of AQP4 expression, we determined whether exogenous LTD4 induces AQP4 expression in the rat primary astrocyte cultures and which subtype of the CysLT receptors is involved in LTL₄-induced up-regulation of AQP4 expression. We found that LTD4 (10^-9-10^-7 M, for 24 h) concentration-dependently enhanced AQP4 expression. The enhanced AQP4 expression was inhibited by Bay u9773, a non-selective CysLT₁/CysLT₂ receptor antagonist, but not by pranlukast. LTD₄ (10^-9-10^-7M) also induced the mRNA expression of CysLT₂ (not CysLT₁) receptor mRNA in the astrocytes. These results indicate that CysLT₂ receptor may mediate AQP4-related cytotoxic edema and contribute to the brain injury induced by CysLTs.Summary1. Exogenous CysLTs (LTC₄ and LTD₄) induce cortical injury in dose- and time-dependent manners, which relates to blood-brain barrier (BBB) disruption and brain injury. The CysLT₁ receptor antagonist pranlukast protects against BBB disruption and cortical injury. These findings indicate that CysLTs promote the CysLT₁ receptor-mediated formation of vasogenic brain edema and brain injury.2. Exogenous LTD4 enhanced cortical AQP4 expression and brain edema; pranlukast does not inhibit LTT₄-induced AQP4 up-regulation and brain edema.3. Exogenous LTD4 concentration-dependently enhanced AQP4 expression in the rat primary astrocyte cultures. The CysLT₁ receptor antagonists, pranlukast and montelukast, do not inhibit LTD₄-induced up-regulation of AQP4 expression; however, the non-selective CysLT receptor antagonist BAY u9773 inhibited this up-regulation in a concentration-dependent manner. LTD4 also concentration-dependently induces the expression of CysLT₂ receptor mRNA. These findings indicate that CysLTs may promote the cytotoxic brain edema via CysLT₂ receptor-mediated AQP4 up-regulation.