Cysteinyl Leukotriene Receptors Modulate Mouse Brain Excitotoxic Injury And Cryoinjury

Cerebral ischemia and traumatic brain injury are common central nervous diseases with high mortality and disability. Exploring the mechanisms under injury/anti-injury of cerebral ischemia and traumatic brain injury is very important for the treatment of such diseases. Over the past 15-20 years, substantial evidence has accumulated implicating excitotoxicity in the pathogenesis of ischemic brain injury. Excitotoxicity is mainly mediated by a crucial glutamate receptor, N-methyl-D-aspartate(NMDA) receptor. NMDA receptor antagonists can reduce the neuronal death in animal models of ischemic brain injury.On the other hand, 5-lipoxygenase (5-LOX) is a key enzyme metabolizing arachidonic acid to form inflammatory mediators, leukotriene B₄ (LTB₄) and cysteinyl leukotrienes (CysLTs, including LTC₄, LTD₄ and LTE₄). CysLTs are potent inflammatory mediators and involved in various diseases. The actions of CysLTs, such as smooth muscle contraction, microvascular leakage and other pathophysiological changes, are mediated via activating their receptors. Two CysLT receptors have been identified and cloned, namely CysLT₁ and CysLT₂ receptors. Both of them are classical Gq protein-coupled seven transmembrane proteins. A lot ofCysLT receptor antagonists have been discovered, such as montelukast and pranlukast. In early studies, CysLT₁ and CysLT₂ receptors were initially found in peripheral organs. Therefore, most studies focused on their roles in peripheral diseases, such as the diseases of respiratory system. In recent studies, CysLT₁ and CysLT₂ receptors are reported to be also expressed in the brain, and they may be involved in the pathogenesis of various central nervous diseases, such as cerebral ischemia, brain trauma and aging. After cerebral ischemia, both the expression and enzymatic activity of 5-LOX in the brain are increased, and the contents of CysLTs are elevated in the brain tissue. Furthermore, the expression of CysLT₁ and CysLT₂ receptors can be induced after cerebral ischemia.Our previous studies have also shown the protective effect of CysLT₁ receptor antagonists, pranlukast and montelukast, on experimental cerebral ischemia. We have recently reported that CysLT₁ and CysLT₂ receptor expressions are induced in the neuron- and glial-appearing cells after traumatic injury in human brain specimens. For the relation between CysLTs and excitotoxicity, it has previously reported that CysLTs levels are increased after cerebral ischemia and this formation is associated with NMDA receptor activation. However, no direct evidence shows the role of CysLT₁ and CysLT₂ receptors in the excitotoxic injury. Are CysLT₁ and CysLT₂ receptors expressed in neurons? Are they involved in N-methyl-D-aspartate-mediated neuronal injury? These questions should be clarified.Therefore, in the present study, we want to answer the above questions by establishing an in vivo model of excitotoxicity injury induced by direct NMDA microinjection into the mouse cerebral cortex. We also observed the changes of CysLT₁ receptor expression in another brain injury model -mouse brain cryoinjury, and further determined the effect of CysLT₁ receptor selective antagonist, pranlukast, after administered after cryoinjury.Part 1 Cysteinyl leukotriene receptor 1 is involved in N-methyl-D-aspartate-mediated neuronal injury in miceExcitotoxicity plays a determinant role in brain injury. Cysteinyl leukotrienes (CysLTs), potent inflammatory mediators, and their CysLT₁ receptor are also involved in brain injury. We recently reported that an inducible expression of CysLT₁ receptor was found in neuron- and glial-appearing cells after traumatic injury in human brain. To determine whether CysLT₁ receptor is involved in N-methyl-D-aspartate (NMDA)-induced excitotoxic injury in mouse brain, we established brain excitotoxic injury model by NMDA microinjection (50-150 nmol in 0.5 μl\) into the cerebral cortex. The changes in CysLT₁ receptor expression as well as the effects of the CysLT₁ receptor antagonist pranlukast (0.01 and 0.1 mg/kg), the NMDA receptor antagonist ketamine (30 mg/kg) and the antioxidant edaravone (9 mg/kg) were observed 24 h after NMDA injection. We found that CysLT₁ receptor mRNA and protein expression were up-regulated and mainly localized in neurons. Pranlukast, ketamine and edaravone decreased NMDA-induced injury; pranlukast (0.1 mg/kg) and ketamine inhibited the up-regulated expression of CysLT₁ receptor. Therefore, the expression of CysLT₁ receptor in the neurons is up-regulated after exposure to NMDA; NMDA-induced responses are inhibited by CysLT₁ receptor antagonist. These findings indicate that the increased CysLT₁ receptor is involved in NMDA excitotoxicity.Part 2 CysLT₂ receptor expression in mouse brain after N-methyl-D-aspartate-mediated neuronal injuryCysLT₂ receptor is one of the two CysLT receptors cloned. It is highly expressed in human brain and distributed in most regions of the brain. We recently reported an inducible expression of CysLT₂ receptor in neuron- and glial-appearing cells after traumatic injury in human brain. To clarify the role of CysLT₂ receptor in N-methyl-D-aspartate (NMDA)-mediated neuronal injury, we investigated the expression of CysLT₂ receptor in mouse brain after NMDA microinjection into the cerebral cortex using RT-PCR and immunohistochemisty. We found that in the NMDA -injured brain, the expression of CysLT₂ receptor mRNA was increased 24 h after NMDA microinjection. CysLT₂ receptor protein was primarily localized in the neurons after NMDA insult as detected by immunohistochemisty. These findings indicate that CysLT₂ receptor may be involved in NNDA-mediated neuronal injury.Part 3 CysLT₁ receptor expression in mouse brain after cryoinjury and time-dependent effect of CysLT₁ receptor antagonist pranlukastWe recently reported an inducible expression of CysLT₁ receptor was found in neuron- and glial-appearing cells after traumatic injury in human brain. To further determine the role of CysLT₁ receptor in traumatic brain injury (TBI), we induced brain cryoinjury (a TBI model) by using metal probe that was previously cooled in liquid nitrogen. The changes in CysLT₁ receptor expression in mouse brain was observed from 3 h to 48 h after cryoinjury, and the time-dependent effects of CysLT₁ receptor antagonist pranlukast (0.01 and 0.1 mg/kg) and minocycline (45 mg/kg) were evaluated. We found that CysLT₁ receptor mRNA expression was up-regulated in the cryoinjured brain from 6 h to 24 h; the up-regulated CysLT₁ receptor protein was primarily localized in the neurons, not in the astrocytes. Post-injury administration of pranlukast (0.1 mg/kg) decreased the injuries with a therapeutic window of 30 min. The positive control, minocycline, decreased the cryoinjury and also inhibited the up-regulation of the CysLT₁ receptor expression. Thus, CysLT₁ receptor mightmediate acute neuronal damage after brain cryoinjury; CysLT₁ receptor antagonist, pranlukast, has a therapeutic potential in the treatment of brain traumatic injury.Conclusions1. The expressions of CysLT₁ and CysLT₂ receptors in mouse brain are up-regulated after NNDA-induced excitotoxic injury. CysLT₁ and CysLT₂ receptors are primarily localized in the neurons, but not in astrocytes, in the injury region 24 h after NMDA microinjection into mouse cortex. Therefore, both CysLT₁ and CysLT₂ receptors are involved in NMDA-induced excitotoxicity.2. CysLT₁ receptor antagonist, pranlukast, attenuates the neuronal damage and inhibits the CysLT₁ receptor expression after NMDA injury. These findings further confirm that CysLT₁ receptor mediates acute neuronal damage after NMDA-induced excitotoxicity.3. The expression of CysLT₁ receptor in mouse brain is up-regulated after brain cryoinjury with peaks at 6-24 h. CysLT₁ receptor are primarily localized in the neurons in the periphery of the lesion. CysLT₁ receptor antagonist, pranlukast, time-dependently protects against brain cryoinjury and its therapeutic window is 30 min after injury. These findings indicate that CysLT₁ receptor might mediate acute neuronal damage after cryoinjury and pranlukast has a therapeutic potential in the treatment of brain traumatic injury.