Shotaro Michinaga

Pathogenesis of Brain Edema and Investigation into Anti-Edema Drugs

Brain edema is a potentially fatal pathological state that occurs after brain injuries such as stroke and head trauma. In the edematous brain, excess accumulation of extracellular fluid results in elevation of intracranial pressure, leading to impaired nerve function. Despite the seriousness of brain edema, only symptomatic treatments to remove edema fluid are currently available. Thus, the development of novel anti-edema drugs is required. The pathogenesis of brain edema is classified as vasogenic or cytotoxic edema. Vasogenic edema is defined as extracellular accumulation of fluid resulting from disruption of the blood-brain barrier (BBB) and extravasations of serum proteins, while cytotoxic edema is characterized by cell swelling caused by intracellular accumulation of fluid. Various experimental animal models are often used to investigate mechanisms underlying brain edema. Many soluble factors and functional molecules have been confirmed to induce BBB disruption or cell swelling and drugs targeted to these factors are expected to have anti-edema effects. In this review, we discuss the mechanisms and involvement of factors that induce brain edema formation, and the possibility of anti-edema drugs targeting them.

Endothelins reciprocally regulate VEGF-A and angiopoietin-1 production in cultured rat astrocytes: implications on astrocytic proliferation.

Vascular endothelial growth factors (VEGFs) and angiopoietins (ANGs) are involved in pathophysiological responses in damaged nerve tissues. Astrocytes produce VEGFs and ANGs upon brain ischemia and traumatic injury. To clarify the extracellular signals regulating VEGF and ANG production, effects of endothelins (ETs), a family of endothelium‐derived peptides, were examined in cultured rat astrocytes. ET‐1 (100 nM) and Ala1,3,11,15‐ET‐1 (100 nM), an ETB receptor agonist, increased VEGF‐A mRNA levels in cultured astrocytes, while ANG‐1 mRNA was decreased by ETs. ET‐1 did not affect astrocytic VEGF‐B, placental growth factor (PLGF), and ANG‐2 mRNA levels. The effects of ET‐1 on VEGF‐A and ANG‐1 mRNAs were inhibited by BQ788, an ETB antagonist. Release of VEGF‐A proteins from cultured astrocytes was increased by ET‐1. In contrast, ET‐1 reduced release of astrocytic ANG‐1. Exogenous ET‐1 (100 nM) and VEGF165 (100 ng/mL), an isopeptide of VEGF‐A, stimulated bromodeoxyuridine (BrdU) incorporation into cultured astrocytes. Treatment with ET‐1 and VEGF165 increased the numbers of cyclin D1‐positive astrocytes. Exogenous ANG‐1 (250 ng/mL) did not stimulate the BrdU incorporation. Increases in BrdU incorporation by ET‐1 and VEGF165 were not affected by ANG‐1. In 60–70% confluent cultures, SU4312 (10 μM), a VEGF receptor tyrosine kinase inhibitor, partially reduced the effects of ET‐1 on BrdU incorporation and cyclin D1 expression. ET‐induced BrdU incorporation and cyclin D1 expression were reduced by a neutralizing antibody against VEGF‐A. Our findings suggest that ET‐1 is a factor regulating astrocytic VEGF‐A and ANG‐1, and that increased VEGF‐A production potentiates ET‐induced astrocytic proliferation by an autocrine mechanism.

Different actions of endothelin-1 on chemokine production in rat cultured astrocytes: reduction of CX3CL1/fractalkine and an increase in CCL2/MCP-1 and CXCL1/CINC-1

AbstractBackgroundChemokines are involved in many pathological responses of the brain. Astrocytes produce various chemokines in brain disorders, but little is known about the factors that regulate astrocytic chemokine production. Endothelins (ETs) have been shown to regulate astrocytic functions through ETB receptors. In this study, the effects of ETs on chemokine production were examined in rat cerebral cultured astrocytes.MethodsAstrocytes were prepared from the cerebra of one- to two-day-old Wistar rats and cultured in serum-containing medium. After serum-starvation for 48 hours, astrocytes were treated with ETs. Total RNA was extracted using an acid-phenol method and expression of chemokine mRNAs was determined by quantitative RT-PCR. The release of chemokines was measured by ELISA.ResultsTreatment of cultured astrocytes with ET-1 and Ala1,3,11,15-ET-1, an ETB agonist, increased mRNA levels of CCL2/MCP1 and CXCL1/CINC-1. In contrast, CX3CL1/fractalkine mRNA expression decreased in the presence of ET-1 and Ala1,3,11,15-ET-1. The effect of ET-1 on chemokine mRNA expression was inhibited by BQ788, an ETB antagonist. ET-1 increased CCL2 and CXCL1 release from cultured astrocytes, but decreased that of CX3CL1. The increase in CCL2 and CXCL1 expression by ET-1 was inhibited by actinomycin D, pyrrolidine dithiocarbamate, SN50, mithramycin, SB203580 and SP600125. The decrease in CX3CL1 expression by ET-1 was inhibited by cycloheximide, Ca2+ chelation and staurosporine.ConclusionThese findings suggest that ETs are one of the factors regulating astrocytic chemokine production. Astrocyte-derived chemokines are involved in pathophysiological responses of neurons and microglia. Therefore, the ET-induced alterations of astrocytic chemokine production are of pathophysiological significance in damaged brains.

Amelioration of cold injury-induced cortical brain edema formation by selective endothelin ETB receptor antagonists in mice.

Brain edema is a potentially fatal pathological condition that often occurs in stroke and head trauma. Following brain insults, endothelins (ETs) are increased and promote several pathophysiological responses. This study examined the effects of ETB antagonists on brain edema formation and disruption of the blood-brain barrier in a mouse cold injury model (Five- to six-week-old male ddY mice). Cold injury increased the water content of the injured cerebrum, and promoted extravasation of both Evans blue and endogenous albumin. In the injury area, expression of prepro-ET-1 mRNA and ET-1 peptide increased. Intracerebroventricular (ICV) administration of BQ788 (ETB antagonist), IRL-2500 (ETB antagonist), or FR139317 (ETA antagonist) prior to cold injury significantly attenuated the increase in brain water content. Bolus administration of BQ788, IRL-2500, or FR139317 also inhibited the cold injury-induced extravasation of Evans blue and albumin. Repeated administration of BQ788 and IRL-2500 beginning at 24 h after cold injury attenuated both the increase in brain water content and extravasation of markers. In contrast, FR139317 had no effect on edema formation when administrated after cold injury. Cold injury stimulated induction of glial fibrillary acidic protein-positive reactive astrocytes in the injured cerebrum. Induction of reactive astrocytes after cold injury was attenuated by ICV administration of BQ788 or IRL-2500. These results suggest that ETB receptor antagonists may be an effective approach to ameliorate brain edema formation following brain insults.

An anti-Parkinson drug ropinirole depletes orexin from rat hypothalamic slice culture.

Non-ergot-type dopamine receptor agonists such as ropinirole are used for the treatment of Parkinson disease, but they occasionally show serious side effects including sleep attacks and daytime sleepiness. These symptoms are reminiscent of narcolepsy, a major sleep disorder. Because narcolepsy is thought to result from deficiency of a hypothalamic neuropeptide orexin, we examined whether ropinirole affected the integrity of orexin-containing neurons, using organotypic slice culture of rat hypothalamus. Application of ropinirole induced a significant decrease in the number of orexin-immunoreactive neurons. The same treatment showed no significant effect on the number of melanin-concentrating hormone-immunoreactive neurons. The decrease of orexin-immunoreactive neurons was reversible after washout of ropinirole and was not accompanied by induction of cell death. Antagonism of dopamine D(2) receptors and of serotonin 5-HT(1A) receptors attenuated the effect of ropinirole, suggesting involvement of these receptors in depletion of orexin. On the other hand, a moderate concentration of N-methyl-d-aspartate that excited orexin neurons counteracted the effect of ropinirole on the number of orexin-immunoreactive neurons. These results suggest that ropinirole can cause deficiency of orexin by inhibiting excitatory activities of orexin neurons, which may be relevant to the adverse actions of this drug on sleep and wakefulness.

Orexin neurons in hypothalamic slice cultures are vulnerable to endoplasmic reticulum stress

Narcolepsy results from disruption of orexin neurons in the hypothalamus that play a key role in maintenance of the arousal state. Underlying mechanisms leading to selective loss of orexin neurons remain unknown. On the other hand, endoplasmic reticulum stress, namely, conditions associated with impairment of endoplasmic reticulum functions such as proper folding and sorting of newly synthesized proteins, is implicated in pathogenesis of several types of neurodegenerative disorders. Here we found that application of endoplasmic reticulum stress inducers such as tunicamycin (that prevents protein N-glycosylation) and thapsigargin (that inhibits Ca²⁺-ATPase) to organotypic slice cultures of the hypothalamus caused preferential loss of orexin-immunoreactive neurons, as compared to melanin-concentrating hormone- or calcitonin gene-related peptide-immunoreactive neurons. The decrease in orexin-immunoreactive neurons at early time points (6-24 h) was not accompanied by induction of cell death as indicated by the absence of caspase-3 activation and no significant change in the number of NeuN-positive cells, whereas sustained treatment with tunicamycin for 72 h induced cell death. At 24-h treatment, tunicamycin and thapsigargin did not decrease expression of prepro-orexin mRNA, suggesting that post-transcriptional mechanisms were responsible for depletion of orexin peptides. In addition, inhibition of axonal transport by colchicine and inhibition of proteasomal activity by MG132 significantly prevented the decrease in orexin immunoreactivity by tunicamycin. Comparative examinations of expression of unfolded protein response-related proteins revealed that C/EBP-homologous protein (a transcription factor that promotes induction of apoptosis) as well as phosphorylated form of RNA-dependent protein kinase-like endoplasmic reticulum kinase (a protein kinase that mediates inhibition of protein translation) was expressed more prominently in orexin neurons than in melanin-concentrating hormone neurons, in response to tunicamycin. These results indicate that orexin neurons are particularly sensitive to endoplasmic reticulum stress, which may be relevant to pathogenic events in narcolepsy.

Improvement of cold injury‐induced mouse brain edema by endothelin ETB antagonists is accompanied by decreases in matrixmetalloproteinase 9 and vascular endothelial growth factor‐A

Brain edema is a potentially fatal pathological state that often occurs after brain injuries such as ischemia and trauma. However, therapeutic agents that fundamentally treat brain edema have not yet been established. We previously found that endothelin ETB receptor antagonists attenuate the formation and maintenance of vasogenic brain edema after cold injury in mice. In this study, the effects of ETB antagonists on matrixmetalloproteinase (MMP)9 and vascular endothelial growth factor (VEGF)‐A expression were examined in the cold injury model. Cold injury was performed in the left brain of male ddY mice (5–6 weeks old) for the induction of vasogenic edema. Expression of MMP9 and VEGF‐A mRNA in the mouse cerebrum was increased by cold injury. Immunohistochemical observations showed that the MMP9 and VEGF‐A were mainly produced in reactive astrocytes in the damaged cerebrum. Intracerebroventricular administration of BQ788 (10 μg) or IRL‐2500 (10 μg) (selective ETB antagonists) attenuated brain edema and disruption of the blood–brain barrier after cold injury. BQ788 and IRL‐2500 reversed the cold injury‐induced increases in MMP9 and VEGF‐A expression. The induction of reactive astrocytes producing MMP9 and VEGF‐A in the damaged cerebrum was attenuated by BQ788 and IRL‐2500. These results suggest that attenuations of astrocytic MMP9 and VEGF‐A expression by ETB antagonists may be involved in the amelioration of vasogenic brain edema.

Anti-Parkinson Drugs and Orexin Neurons

Non-ergot-type dopamine receptor agonists such as ropinirole are used for treatment of Parkinson disease, but they frequently produce adverse actions characterized by sleepiness and sleep attacks. Because these symptoms are similar to those observed in patients with narcolepsy, a sleep disorder caused by degeneration of hypothalamic orexin neurons, involvement of orexinergic system in the adverse drug actions is suspected. We found that ropinirole and other non-ergot dopamine D₂ receptor agonists cause selective loss of orexin-immunoreactive neurons in organotypic slice culture of rat hypothalamus. The mechanism of this action is considered to involve D₂ receptor-mediated presynaptic suppression of glutamatergic excitatory inputs to orexin neurons because continuous silencing of excitatory activity of orexin neurons can deplete orexin from cell bodies. In addition, Parkinson disease itself may accompany loss of orexin neurons. Disturbance of orexinergic system may play an important role in sleep/arousal dysfunctions under these and other clinical conditions.

Inhibition of neural activity depletes orexin from rat hypothalamic slice culture.

Orexins (hypocretins) are neuropeptides produced by a small population of hypothalamic neurons whose dysregulation may lead to narcolepsy, a neurological disorder characterized by disorganization of sleep and wakefulness. Excessive stimulation of the N‐methyl‐D‐aspartate (NMDA) subtype of glutamate receptors causes preferential loss of orexin neurons in the hypothalamus, whereas an adequate level of neuronal excitatory activities is generally known to be important for the maintenance of central neurons. By examining the effect of manipulation of neural activity, we found that 24–72 hr application of tetrodotoxin (TTX) caused a substantial decrease in the number of orexin‐immunoreactive neurons, but not of melanin‐concentrating hormone‐immunoreactive neurons, in hypothalamic slice culture. Similar results were obtained when neural activity was arrested by added extracellular Mg2+. Reduction of orexin expression by TTX and Mg2+ was also observed at mRNA level. The decrease of orexin‐immunoreactive neurons was attributable to depletion of orexin, because it was reversible after washout of TTX or elevated extracellular Mg2+ and was not associated with induction of cell death. Blockers of voltage‐dependent Ca2+ channels as well as of NMDA receptors also induced a significant and selective decrease of orexin‐immunoreactive neurons. Moreover, TTX‐induced decrease of orexin immunoreactivity was largely abrogated by concurrent application of a moderate concentration of NMDA. These results suggest that Ca2+ entry associated with nontoxic levels of spontaneous activity of glutamatergic inputs plays an important role in the maintenance of orexin neurons in a tissue culture model.

Endothelin-1 stimulates cyclin D1 expression in rat cultured astrocytes via activation of Sp1

Endothelins (ETs), a family of vasoconstrictor peptides, are up-regulated in several pathological conditions in the brain, and induce astrocytic proliferation. We previously observed that ET-1 increased the expression of cyclin D1 protein. Thus, we confirmed the intracellular up-regulation of cyclin D1 by ET-1 in rat cultured astrocytes. Real-time PCR analysis indicated that ET-1 (100 nM) and Ala(1,3,11,15)-ET-1 (100 nM), a selective agonist of the ETB receptor, induced a time-dependent and transient increase in cyclin D1 mRNA. The effect of ET-1 was diminished by an ETB antagonist (1 μM BQ788) or inhibitors of Sp1 (500 nM mithramycin), ERK (50 μM PD98059), p38 (20 μM SB203580) and JNK (1 μM SP600125), but not inhibitors of NF-κB (10 μM SN50 and 100 μM pyrrolidine dithiocarbamate). The binding assay for Sp1 indicated that ET-1 increased the binding activity of Sp1 to consensus sequences, and two oligonucleotides of the cyclin D1 promoter including the Sp1-binding sites diminished the effect of ET-1. Western blot analysis showed that ET-1 induced time-dependent and transient phosphorylation of Sp1 on Thr453 and Thr739 via the ETB receptor. ET-1-induced phosphorylation of Sp1 was attenuated by PD98059 and SP600125. Additionally, ET-1 increased the incorporation of bromodeoxyuridine (BrdU) in cultured astrocytes and the number of BrdU-positive cells decreased in the presence of PD98059, SP600125 and mithramycin. These results suggest that ET-1 increases the expression of cyclin D1 via activation of Sp1 and induces astrocytic proliferation.