Evaluation of ADMA-DDAH-NOS axis in specific brain areas following nitroglycerin administration: study in an animal model of migraine.

BACKGROUND: Nitric oxide (NO) is known to play a key role in migraine pathogenesis, but modulation of NO synthesis has failed so far to show efficacy in migraine treatment. Asymmetric dimethylarginine (ADMA) is a NO synthase (NOS) inhibitor, whose levels are regulated by dimethylarginine dimethylaminohydrolase (DDAH). Systemic administration of nitroglycerin (or glyceryl trinitrate, GTN) is a NO donor that consistently induces spontaneous-like headache attacks in migraneurs. GTN administration induces an increase in neuronal NOS (nNOS) that is simultaneous with a hyperalgesic condition. GTN administration has been used for years as an experimental animal model of migraine. In order to gain further insights in the precise mechanisms involved in the relationships between NO synthesis and migraine, we analyzed changes induced by GTN administration in ADMA levels, DDHA-1 mRNA expression and the expression of neuronal and endothelial NOS (nNOS and eNOS) in the brain. We also evaluated ADMA levels in the serum. METHODS: Male Sprague-Dawley rats were injected with GTN (10 mg/kg, i.p.) or vehicle and sacrificed 4 h later. Brain areas known to be activated by GTN administration were dissected out and utilized for the evaluation of nNOS and eNOS expression by means of western blotting. Cerebral and serum ADMA levels were measured by means of ELISA immunoassay. Cerebral DDAH-1 mRNA expression was measured by means of RT-PCR. Comparisons between experimental groups were performed using the Mann Whitney test. RESULTS: ADMA levels and nNOS expression increased in the hypothalamus and medulla following GTN administration. Conversely, a significant decrease in DDAH-1 mRNA expression was observed in the same areas. By contrast, no significant change was reported in eNOS expression. GTN administration did not induce any significant change in serum levels of ADMA. CONCLUSION: The present data suggest that ADMA accumulates in the brain after GTN administration via the inhibition of DDAH-1. This latter may represent a compensatory response to the excessive local availability of NO, released directly by GTN or synthetized by nNOS. These findings prompt an additional mediator (ADMA) in the modulation of NO axis following GTN administration and offer new insights in the pathophysiology of migraine.

Intracarotid Infusion of Mesenchymal Stem Cells in an Animal Model of Parkinson's Disease, Focusing on Cell Distribution and Neuroprotective and Behavioral Effects.

UNLABELLED: Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for Parkinson's disease (PD) and systemic administration of these cells has been tested in preclinical and clinical studies. However, no information on survival and actual capacity of MSCs to reach the brain has been provided. In this study, we evaluated homing of intraarterially infused rat MSCs (rMSCs) in the brain of rats bearing a 6-hydroxydopamine (6-OHDA)-induced lesion of the nigrostriatal tract, to establish whether the toxin-induced damage is sufficient to grant MSC passage across the blood-brain barrier (BBB) or if a transient BBB disruption is necessary. The rMSC distribution in peripheral organs and the effects of cell infusion on neurodegenerative process and motor deficits were also investigated. rMSCs were infused 14 days after 6-OHDA injection. A hyperosmolar solution of mannitol was used to transiently permeabilize the BBB. Behavioral impairment was assessed by adjusting step test and response to apomorphine. Animals were sacrificed 7 and 28 days after cell infusion. Our work shows that appreciable delivery of rMSCs to the brain of 6-OHDA-lesioned animals can be obtained only after mannitol pretreatment. A notable percentage of infused cells accumulated in peripheral organs. Infusion of rMSCs did not modify the progression of 6-OHDA-induced damage or the motor impairment at the stepping test, but induced progressive normalization of the pathological response (contralateral turning) to apomorphine administration. These findings suggest that many aspects should be further investigated before considering any translation of MSC systemic administration into the clinical setting for PD treatment. SIGNIFICANCE: This study demonstrates that mesenchymal stem cells infused through the carotid artery do not efficiently cross the blood-brain barrier in rats with a Parkinson's disease-like degeneration of nigrostriatal neurons, unless a permeabilizing agent (e.g., mannitol) is used. The infusion did not reduce the neuronal damage and associated motor impairment, but abolished the motor abnormalities these animals typically show when challenged with a dopaminergic agonist. Therefore, although arterially infused mesenchymal stem cells did not show neurorestorative effects in this study's Parkinson's disease model, they appeared to normalize the pathological responsiveness of striatal neurons to dopaminergic stimulation. This capability should be further explored in future studies.

Neuroprotection by the PARP inhibitor PJ34 modulates cerebral and circulating RAGE levels in rats exposed to focal brain ischemia.

The receptor for advanced glycation end products (RAGE) has a potential role as a damage-sensing molecule; however, to date, its involvement in the pathophysiology of stroke and its modulation following neuroprotective treatment are not completely understood. We have previously demonstrated that expression of distinct RAGE isoforms, recognized by different antibodies, is differentially modulated in the brain of rats subjected to focal cerebral ischemia. Here, we focus on the full-length membrane-bound RAGE isoform, showing that its expression is significantly elevated in the striatum, whereas it is reduced in the cortex of rats subjected to transient middle cerebral artery occlusion (MCAo). Notably, the reduction of cortical levels of full-length RAGE detected 24 h after reperfusion is abolished by systemic administration of a neuroprotective dose of the poly(ADP-ribose) polymerase (PARP) inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34). More interestingly, a significant reduction of plasma soluble RAGE (sRAGE) occurs 24 h after reperfusion and this effect is reverted by a neuroprotective dose of PJ34. Soluble forms of RAGE, generated either by alternative splicing or by proteolysis of the full-length form, effectively bind advanced glycation end products, thereby competing with the cell surface full-length RAGE, thus providing a 'decoy' function that may counteract the adverse effects of receptor signaling in neurons and may possibly exert cytoprotective effects. Thus, our data confirm the important role of RAGE in ischemic cerebral damage and, more interestingly, suggest the potential use of sRAGE as a blood biomarker of stroke severity and of neuroprotective treatment efficacy.

Activation of CB2 receptors as a potential therapeutic target for migraine: evaluation in an animal model.

BACKGROUND: Experimental animal models of migraine have suggested the existence of interactions between the endocannabinoid system and pain mediation in migraine. Extensive evidence has demonstrated a role for the cannabinoid-1 (CB1) receptor in antinociception. However, recent research suggests that also CB2 receptors, especially located outside the central nervous system, play a role in the perception of pain. Systemic administration of nitroglycerin (NTG) consistently induces spontaneous-like headache attacks in migraneurs; in the rat, systemic NTG induces a condition of hyperalgesia, probably through the activation of cerebral/spinal structures involved in nociceptive transmission. In this study we evaluated the role of CB2 receptors in two animal models of pain that may be relevant for migraine: the tail flick test and the formalin test performed during NTG-induced hyperalgesia. METHODS: The study was performed in male Sprague-Dawley rats pre-treated with NTG (10 mg/kg, i.p.) or vehicle (4 hours before) and treated with the CB2 agonist AM1241 o dimethylsulfoxide (DMSO) 60 minutes before both the tail flick test and the formalin test. RESULTS: AM1241 showed a significant analgesic effect in baseline conditions in both tests. Furthermore, when administered 3 hours after NTG administration, AM1241 at both doses significantly reduced the total number of flinches/shakes during phase II of the test. CONCLUSION: These findings suggest that the pharmacological manipulation of the CB2 receptor may represent a potential therapeutic tool for the treatment of migraine.

Modulation of RAGE isoforms expression in the brain and plasma of rats exposed to transient focal cerebral ischemia.

Activation of RAGE (receptor for advanced glycation endproducts) and of its subtypes may play a role in neuronal damage and neuroinflammation associated with brain ischemia, though the underlying mechanisms remain unclear. In this study, we have examined by Western blotting the expression of RAGE isoforms in the cerebral cortex and striatum of Wistar rats subjected to transient (1 or 2 h) middle cerebral artery occlusion (tMCAo). The findings show that the full-length RAGE (~50 kDa) and its isoforms in the 26-43 kDa range are significantly decreased in the ischemic cortex, but not in the striatum, after 1 and 2 h tMCAo when compared to the sham group. By contrast, in the striatum, ischemia-reperfusion injury caused a significant increase of full-length RAGE and its isoforms in the 72-100 kDa range. We also investigated the soluble form of RAGE, which was significantly decreased in the plasma of rats subjected to transient or permanent MCAo. In conclusion, the present data demonstrate that regional brain expression of RAGE is differentially affected by tMCAo in rat. These modifications are accompanied by a decrease in the plasma levels of soluble RAGE, thereby suggesting a potential role for soluble RAGE as a peripheral biomarker of focal ischemia.

Effects of anandamide in migraine: data from an animal model.

Systemic nitroglycerin (NTG) produces spontaneous-like migraine attacks in migraine sufferers and induces a condition of hyperalgesia in the rat 4 h after its administration. Endocannabinoid system seems to be involved in the modulation of NTG-induced hyperalgesia, and probably, in the pathophysiological mechanisms of migraine. In this study, the analgesic effect of anandamide (AEA) was evaluated by means of the formalin test, performed in baseline conditions and following NTG-induced hyperalgesia in male Sprague-Dawley rats. AEA was administered 30 min before the formalin injection. In addition, the effect of AEA (administered 30 min before NTG injection) was investigated on NTG-induced Fos expression and evaluated 4 h following NTG injection. AEA induced a significant decrease in the nociceptive behavior during both phases of the formalin test in the animals treated with vehicle, while it abolished NTG-induced hyperalgesia during the phase II. Pre-treatment with AEA significantly reduced the NTG-induced neuronal activation in nucleus trigeminalis caudalis, confirming the results obtained in our previous study, and in area postrema, while the same treatment induced an increase of Fos expression in paraventricular and supraoptic nuclei of the hypothalamus, parabrachial nucleus, and periaqueductal grey. The study confirms that a dysfunction of the endocannabinoid system may contribute to the development of migraine attacks and that a pharmacological modulation of CB receptors can be useful for the treatment of migraine pain.

IkappaB-alpha expression following transient focal cerebral ischemia is modulated by nitric oxide.

The role of nitric oxide (NO) in cerebral ischemia/reperfusion (IR) has been intensively investigated. In general NO is regarded as a mediator of ischemia-associated neuronal damage, as inhibitors of NO synthesis ameliorate neuronal injury during permanent focal cerebral ischemia, however the exact role of NO in ischemia remains controversial. It has been previously shown that NO-donors can directly inhibit the DNA binding activity of NF-kappaB family proteins and strong evidence supports that activation of NF-κB contributes to ischemia-induced neuronal injury. In this study, we have investigated whether NO production by nNOS, eNOS and iNOS modulates IkB-alpha expression in cerebral ischemia, by using various inhibitors of NOS, in rats subjected to transient (1h) middle cerebral artery occlusion (tMCAo). Male Wistar rats were treated intraperitoneally (i.p.) with 3mg/kg of NG-nitro-l-arginine methyl ester (l-NAME, a non-selective NOS inhibitor), 5mg/kg of l-N6-(1-iminoethyl)-lysine (l-NIL, an inducible NOS inhibitor), 25mg/kg of 7-Nitroindazole (7-NI, a neuronal NOS inhibitor) and 10mg/Kg of l-N-(1-iminoethyl)ornithine (l-NIO, a selective eNOS inhibitor) 15min before the induction of tMCAo. Cortical IkB-alpha expression was evaluated by western blotting and its decrease was considered as an indicator of NF-κB activation. IkB-alpha expression decreased in ischemic cortices when compared with the cortices of the sham group, thus confirming the activation of NF-κB in ischemic conditions. Pre-treatment with l-NAME, l-NIL and 7-NI significantly reduced the infarct volume and prevented ischemia-induced reduction in IkB-alpha expression. Conversely, pretreatment with l-NIO was associated with a significant increase in infarct volume and a reduction in IkB-alpha expression. These findings suggest that NO of neuronal and inducible origin promotes NF-κB activation via IkB-alpha modulation and mediates ischemic-related damage in the brain following ischemia.

Temporal profile of vascular changes induced by systemic nitroglycerin in the meningeal and cortical districts.

BACKGROUND: Clinical studies indicated that nitric oxide (NO) donors cause regional changes in cerebral blood flow (CBF), similar to those reported in spontaneous migraine. Systemic nitroglycerin (NTG), a NO donor, is a well-accepted experimental model of migraine. In this study we have examined the effects of NTG on the meningeal and cortical blood flow in rats. METHODS: Regional blood flow was monitored in male Sprague-Dawley rats using laser Doppler flowmetry before and after NTG/saline injection over 150 minutes. The effect of pre-treatment with Nω-nitro-L-arginine ester (L-NAME) or 7-nitroindazole (7-NI) on NTG-induced changes on blood flow was also investigated. RESULTS: In the dura NTG caused a biphasic response represented by an initial decrease in blood flow followed by a significant increase. At variance, in the cortex NTG caused only an increase in blood flow. Pre-treatment with either L-NAME or 7-NI prevented NTG-induced increase in blood flow in both districts, while only L-NAME also prevented NTG-induced decrease in dural blood flow. CONCLUSION: The present findings provide additional information on the timing of effects of NTG on blood flow at both the meningeal and cortical levels. These effects seem to be related to vasoregulatory mechanisms and/or metabolic activity in response to the synthesis of endogenous NO.