The Roles Of Cysteinyl Leukotriene Receptors In Brain Ischemic Injury

Cerebral ischemia triggers inflammation reactions in the brain by inducing the expression of cytokines, adhesion molecules, and other inflammatory mediators, and the reactions progress and last for several days. Interventions that attenuate the responses of inflammatory mediators reduce ischemic brain damage, with a potentially wide theropeutic window. 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.CysLTs' actions are mediated via activating their receptors (CysLT receptor). Two subtypes (CysLT₁ and CysLT₂ receptors) of the receptors have been cloned and characterized recently. They are classic G protein-coupled receptors with seven transmembrane domains. Currently, most of CysLT receptor antagonists are CysLT₁receptor selective antagonists, but no CysLT2 receptor selective antagonist is avialable. Therefore, the function of CysLTi receptor is well clarified, and that of CysLT2 receptor is little known. Most studies of CysLTi receptor focus on disorders in peripheral organs, such as respiratory system, but the function of CysLT receptors in central nervous system is not completely investigated. CysLTi receptor is weakly expressed in human brain and CysLT2 receptor is relatively higher expressed in the brain. However, CysLTi and CysLT2 receptor expressions are induced in the neuron- and glia-appearing cells after traumatic injury in human brain specimens. The findings suggest that CysLTi and CysLT2 receptors may play a similar role in brain injury. In addtion, we have also found that CysLTi receptor antagonists (pranlukast and montelukast) protect against cerebral ischemic injury in rats and mice, pharmacologically suggesting that CysLTi receptors are related to cerebral ischemic injury. However, no direct evidence shows the exact localization of CysLTi and CysLT2 receptors in the brain and the temporal and spatial changes after cerebral ischemia.Ischemic brain injury is a highly complex process. One of the acute injuries after cerebral ischemia is the death (necrosis and apoptosis) of neurons. One of the chronic injuries after cerebral ischemia is the formation of a glial scar that results from reactive gliosis (mainly consisted of astrocytes). It is known that CysLTs/CysLT receptor mediates neuronal damage, and is involved in blood-brain barrier (BBB) dysfunction and brain edema. However, direct evidence that CysLT receptors mediate neuronal damage after cerebral ischemia is lack. On the other hand, in the cultured astrocytes, CysLTs can increase the cell proliferation, and CysLTi receptor is pharmacologically related to the gliosis after cerebral ischemia. Also, no direct evidence is available for the role of CysLTi and CysLT2 receptors in gliosis after focal cerebral ischemia in vivo.To clarify the role of CysLT receptor in the brain after focal cerebral ischemia, in the present study, we investigated the temporal and spatial expression of CysLTi and CysLT2 receptors in rat brain from 3 h to 14 days after 30 min of middle cerebral artery occlusion. We also observed the effect of pranlukast, a CysLTi receptor antagonist, on acute and subacute/chronic injury after focal cerebral ischemia.PartiIncreased expression of CysLTi receptor in the brain mediates neuronaldamage and astrogliosis after focal cerebral ischemia in ratsWe recently reported that CysLTi receptor antagonists protected against cerebral ischemic injury, and an inducible expression of CysLTi receptor was found in neuron-and glial-appearing cells after traumatic injury in human brain. To determine the role of CysLTi receptor in ischemic brain injury, we investigated the temporal and spatial expression of CysLTi receptor in rat brain from 3 h to 14 days after 30 min of middle cerebral artery occlusion, and observed the effect of pranlukast, a CysLTi receptor antagonist, on the ischemic injury. We found that CysLTi receptor mRNA expression was up-regulated in the ischemic core both 3-12 h and 7-14 days, and in the boundary zone 7-14 days after reperfusion. In the ischemic core, CysLTi receptor was primarily localized in neurons 24 h, and in macrophage/microglia 14 days after reperfusion;while it was localized in proliferated astrocytes in the boundary zone 14 days after reperfusion. Pranlukast attenuated neurological deficits, reduced infarct volume and ameliorated neuron loss in the ischemic core 24 h after reperfusion;it reduced infarct volume, ameliorated neuron loss and inhibited astrocyte proliferation in the boundary zone 14 days after reperfusion. In addition, immunohistochemistry showed CysLTi receptor was expressed in choroid plexus and endothelial cells in normal rat brain. Thus, we conclude that CysLTi receptor mediates acute neuronal damage and subacute/chronic astrogliosis after focal cerebral ischemia.Part IITemporal and spatial profile of CysLT2 receptor expression in rat brainafter focal cerebral ischemiaCysLT2 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 aninducible expression of CysLT2 receptor that was found in neuron- and glial-appearing cells after traumatic injury in human brain. To clarify the role of CysLT2 receptor in cerebral ischemic injury, we investigated the temporal and spatial expression of CysLT2 receptor in rat brain from 3 h to 14 days after 30 min of middle cerebral artery occlusion using RT-PCR and immunohistochemisty. We found that in the ischemic core the expression of CysLT2 receptor mRNA was increased 6 h, 12 h and 24 h, then recovered 3, 7 and 14 days after reperfusion;in the boundary zone, the expression of CysLT2 receptor mRNA was significantly increased 3, 7 and 14 days after reperfusion. In the ischemic core, CysLT2 receptor was primarily localized in the neurons 24 h after reperfusion;while it was localized in proliferated astrocytes in the boundary zone 14 days after reperfusion. In addition, immunohistochemistry showed that CysLT2 receptor was expressed in the ependymal cells, neurons and astroglial cells around ventricle, especially in thalamus and hypothalamus near ventricle. These findings indicate that CysLT2 receptor may be involved in acute neuronal damage and subacute/chronic astrogliosis after focal cerebral ischemia, and may relate to cerebralspinal fluid circulation and neuroendocrinal modulation under physiological conditions as well.Summary1. The expressions of CysLTi and CysLT2 receptors in rat brain are up-regulated after focal cerebral ischemia. In the ischemic core, CysLTi receptor expression has two peaks at 3-12 h and 7-14 days, respectively, while CysLT2 receptor expression has only one peak at 6-24 h. In the boundary zone adjacent to the ischemic core, both CysLTi and CysLT2 receptor expressions have one peak at 7-14 days for CysLTi receptor or at 3-14 days for CysLT2 receptor.2. Double immunostaining shows that CysLTi and CysLT2 receptors are primarily localized in the neurons in the ischemic core 24 h after reperfusion;while they are localized in the proliferated astrocytes in the boundary zone 14 days after reperfusion. In addition, CysLTi receptor is also localized in macrophage/microglia in the ischemic core 14 days after reperfusion.3. CysLTi receptor antagonist pranlukast attenuates the neuronal damage and inhibits the astrocyte proliferation. These findings further confirm that CysLTi receptor mediates acute neuronal damage and subacute/chronic astrogliosis after focal cerebral ischemia. CysLT2 receptor may be also mediate acute neuronal damage and subacute/chronic astrogliosis;but this mediation needs to be clarified.4. In normal rat brain, CysLTi receptor is expressed in the choroid plexus and endothelial cells. CysLT2 receptor is expressed in the ependymal cells, neurons and astroglial cells around ventricle, especially in thaJamus and hypothalamus near to ventricle. These findings indicate that both CysLTi and CysLT2 receptors may relate to cerebralspinal fluid circulation, and CysLT2 receptor may be involved in neuroendocrinal modulation.