Effect of probenecid on the pain-related behaviour and morphological markers in orofacial formalin test of the rat.

Probenecid has been widely used in the treatment of gout, but evidence suggests that it may also have antinociceptive effects in different inflammatory and pain conditions. We examined the potential modulatory effects of probenecid on behavioural and morphological markers in the orofacial formalin test of the rat. One hour after pre-treatment with vehicle or probenecid (1 mmol/kg body weight) intraperitoneally, 50µl 1.5% formalin solution or physiological saline was injected subcutaneously into the right whisker pad of rats. The rubbing activity directed to the injected whisker pad was then measured for a period of 45 minutes. Four hours after formalin injection, the caudal part of spinal trigeminal nucleus was removed and subjected to c-Fos and neuronal nitric oxide synthase (nNOS) immunohistochemistry and to interleukin-1ß and NAD(P)H: quinone oxidoreductase 1 (NQO1) Western blot. There was a significant decrease in formalin-induced biphasic behavioural response and c-Fos and nNOS immunoreactivity in the rats that were pre-treated with probenecid. However there were no alterations in expression of interleukin-1ß or NQO1 after formalin administration. Our results suggest that probenecid has an anti-nociceptive effect in the trigeminal inflammatory pain model. This effect may be through influencing the release of prostaglandin E2 or desensitizing the transient receptor potential channel subtype A member 1 or the transient receptor potential channel subtype V member 2 or the effect may be through modulating kynurenic acid levels in the central nervous system. Thus, probenecid might be a potential candidate for the treatment of trigeminal activation related pain conditions.

Pre-treatment with new kynurenic acid amide dose-dependently prevents the nitroglycerine-induced neuronal activation and sensitization in cervical part of trigemino-cervical complex.

The systemic administration of nitroglycerine induces attacks in migraineurs and is able to activate and sensitize the trigeminal system in animals involving glutamate and α7-nicotinic acetylcholine receptors, among others. Kynurenic acid is one of the endogenous glutamate receptor antagonists, and exerts inhibitory action on the α7-nicotinic acetylcholine receptors. Since kynurenic acid penetrates the blood-brain barrier poorly, therefore a newly synthesized kynurenic acid amide, N-(2-N-pyrrolidinylethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride (KYNAa) was used with such a side-chain substitution to facilitate brain penetration in our study. We evaluated its modulatory effect on kynurenic acid concentration in the cervical part of trigemino-cervical complex (C1-C2) and in the model of nitroglycerine-induced trigeminal activation using male Sprague-Dawley rats. One hour after 1 mmol/kg bodyweight KYNAa administration, the kynurenic acid level increased significantly in C1-C2, which returned to the basal level at 300 min measured by high-performance liquid chromatography. KYNAa pre-treatment had dose-dependent, mitigating action on nitroglycerine-induced decrease in calcitonin gene-related peptide and increase in c-Fos, neuronal nitric oxide synthase and calmodulin-dependent protein kinase II alpha expression in the C1-C2. KYNAa also mitigated the behavioural changes after nitroglycerine. Thus, in this model KYNAa is able to modulate in a dose-dependent manner the changes in neurochemical markers of activation and sensitization of the trigeminal system directly and indirectly--via forming kynurenic acid, possibly acting on peripheral and central glutamate or α7-nicotinic acetylcholine receptors. These results suggest that application of kynurenic acid derivatives could be a useful therapeutic strategy in migraine headache in the future with a different mechanism of action.

Digital PCR to determine the number of transcripts from single neurons after patch-clamp recording.

Whole-cell patch-clamp recording enables detection of electrophysiological signals from single neurons as well as harvesting of perisomatic RNA through the patch pipette for subsequent gene expression analysis. Amplification and profiling of RNA with traditional quantitative real-time PCR (qRT-PCR) do not provide exact quantitation due to experimental variation caused by the limited amount of nucleic acid in a single cell. Here we describe a protocol for quantifying mRNA or miRNA expression in individual neurons after patch-clamp recording using high-density nanocapillary digital PCR (dPCR). Expression of a known cell-type dependent marker gene (gabrd), as well as oxidative-stress related induction of hspb1 and hmox1 expression, was quantified in individual neurogliaform and pyramidal cells, respectively. The miRNA mir-132, which plays a role in neurodevelopment, was found to be equally expressed in three different types of neurons. The accuracy and sensitivity of this method were further validated using synthetic spike-in templates and by detecting genes with very low levels of expression.

Effect of systemic kynurenine on cortical spreading depression and its modulation by sex hormones in rat.

BACKGROUND: The aura symptoms in migraine are most likely due to cortical spreading depression (CSD). CSD is favored by NMDA receptor activation and increased cortical excitability. The latter probably explains why migraine with aura may appear when estrogen levels are high, like during pregnancy. Kynurenic acid, a derivative of tryptophan metabolism, is an endogenous NMDA receptor antagonist whose cerebral concentrations can be augmented by systemic administration of its precursor L-kynurenine. OBJECTIVE: To determine if exogenous administration of L-kynurenine is able to influence KCl-induced CSD in rat, if the effect is sex-dependent and if it differs in females between the phases of the estrous cycle. METHODS: Adult Sprague-Dawley rats (n=8/group) received intraperitoneal (i.p.) injections of L-kynurenine (L-KYN, 300 mg/kg), L-KYN combined with probenecid (L-KYN+PROB) that increases cortical concentration of KYNA by blocking its excretion from the central nervous system, probenecid alone (PROB, 200 mg/kg) or NaCl. Cortical kynurenic acid concentrations were determined by HPLC (n=7). Thirty minutes after the injections, CSDs were elicited by application of 1M KCl over the occipital cortex and recorded by DC electrocorticogram. In NaCl and L-KYN groups, supplementary females were added and CSD frequency was analyzed respective to the phases of the estrous cycle determined by vaginal smears. RESULTS: In both sexes, PROB, L-KYN and L-KYN+PROB increased cortical kynurenic acid level. PROB, L-KYN and L-KYN+PROB with increasing potency decreased CSD frequency in female rats, while in males such an effect was significant only for L-KYN+PROB. The inhibitory effect of L-KYN on CSD frequency in females was most potent in diestrus. CONCLUSION: L-Kynurenine administration suppresses CSD, most likely by increasing kynurenic acid levels in the cortex. Females are more sensitive to this suppressive effect of L-kynurenine than males. These results emphasize the role of sex hormones in migraine and open interesting novel perspectives for its preventive treatment.

Kynurenate derivative attenuates the nitroglycerin-induced CamKIIα and CGRP expression changes.

OBJECTIVE: To examine the efficacy of L-kynurenine and a novel kynurenic acid derivative on the nitroglycerin-induced calmodulin-dependent protein kinase II alpha (CamKIIalpha) and calcitonin gene-related peptide (CGRP) expression changes in the rat caudal trigeminal nucleus. BACKGROUND: Systemic administration of the nitric oxide donor nitroglycerin can trigger an attack in migraineurs. In the rat, nitroglycerin activates second-order neurons in the caudal trigeminal nucleus, and increases expression of the CamKIIalpha and decreases that of the CGRP there. As glutamatergic mechanisms may be crucial in trigeminal pain processing, the aim of our study was to examine the effects of L-kynurenine, a metabolic precursor of the N-methyl D-aspartate receptor antagonist kynurenic acid, on the nitroglycerin-induced changes in CamKIIalpha and CGRP immunoreactivity. METHODS: One hour before the nitroglycerin (10 mg/kg bodyweight, s.c.) injection, the animals were pretreated with L-kynurenine (300 mg/kg bodyweight, i.p.) or 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1H-quinolin-4-one hydrochloride (300 mg/kg bodyweight, i.p.), a novel kynurenic acid derivative. Four hours later, the rats were perfused transcardially and the cervical spinal cord segments were removed for immunohistochemistry. Results.- L-kynurenine and 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1H-quinolin-4-one hydrochloride pretreatment attenuated the nitroglycerin-induced changes in CamKIIalpha and CGRP immunoreactivity in the rat caudal trigeminal nucleus. CONCLUSIONS: These findings suggest a mechanism by which the inhibition of the excitatory amino acid receptors by kynurenic acid and its derivatives can alter trigeminal nociception.

Neuroprotective effects of L-carnitine in a transgenic animal model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant inherited disorder, caused by an expanded polyglutamine region of a protein called huntingtin. The excitotoxicity, oxidative damage, mitochondrial dysfunction and altered membrane transport may have important roles in the pathogenesis of HD. L-carnitine plays a role in facilitating the mitochondrial transport of fatty acids, but it also protects the cells from oxidative damage. The aim of our study was to examine the effects of the intraperitoneally administered L-carnitine on the survival, behaviour and immunohistochemical changes in the N171-82Q transgenic mouse model of HD. Following L-carnitine administration the survival was improved by 14.9%. The motor activity was significantly ameliorated as compared with the control transgenic group. The L-carnitine treatment significantly reduced the neuronal loss and the number of neuronal intranuclear aggregates. These results suggest that L-carnitine may exert a neuroprotective effect by decreasing the oxidative damage.

Modulatory effects of probenecid on the nitroglycerin-induced changes in the rat caudal trigeminal nucleus.

Four hours after systemic administration of the nitric oxide donor nitroglycerin (10 mg/kg bodyweight, s.c.), the neurons of the rat caudal trigeminal nucleus are activated, the area covered by calcitonin gene-related peptide (CGRP)-immunoreactive fibres is decreased and the neuronal nitric oxide synthase (nNOS)- and the calmodulin-dependent protein kinase II alpha (CamKIIalpha)-immunopositive neurons in the same area are increased. Probenecid is a non-selective inhibitor of multidrug-resistance associated proteins and organic anion transporters thus it can modulate the transport functions in the central nervous system influencing nociception. Accordingly, the aim of the present experiments was to examine the effects of probenecid administration on the nitroglycerin-induced expressions of nNOS, CamKIIalpha and CGRP in the rat caudal trigeminal nucleus. Probenecid (200 mg/kg bodyweight, i.p.) pretreatment proved to mitigate the nitroglycerin-induced changes in expression in the rat caudal trigeminal nucleus. The data suggest that the changes caused by nitroglycerin in the expressions of CGRP, nNOS and CamKIIalpha can be influenced by probenecid modulating the inflammatory functions in the nervous system. These data may be of relevance for the pathogenesis of migraine headache.

Valproate ameliorates the survival and the motor performance in a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is one of the chronic devastating neurodegenerative disorders. The pathophysiological processes clearly involve both excitotoxicity and reduced gene transcription due to the decreased level of histone acetylation, accompanied by the loss of gamma-aminobutyric acidergic (GABAergic) medium-sized spiny neurons in the striatum as a pathological hallmark of HD. Thus, the antiepileptic drug valproate, which has proved GABAergic, antiexcitotoxic and histone deacetylase inhibitor effects, might be of value by exerting a beneficial neuroprotective effect. We have now tested this drug in the N171-82Q transgenic mouse model of HD, following its chronic intraperitoneal administration in a daily dose of 100 mg/kg. Valproate significantly prolonged the survival of the transgenic mice and significantly ameliorated their diminished spontaneous locomotor activity, without exerting any noteworthy side-effect on their behaviour or the striatal dopamine content at the dose administered. The beneficial effect of valproate is probably explained by its complex pharmacological activity. As several previous clinical trials carried out with valproate did not indicate any positive effect in HD, it is worth considering the design of new studies based on a well-planned treatment regime with higher dose, using valproate in monotherapy or in combination therapy with a high number of participating patients.

l-kynurenine combined with probenecid and the novel synthetic kynurenic acid derivative attenuate nitroglycerin-induced nNOS in the rat caudal trigeminal nucleus.

Systemic administration of the nitric oxide (NO) donor nitroglycerin (NTG) triggers a delayed attack without aura in many migraineurs, but not in healthy volunteers. In rats, 4 h after the systemic administration of NTG (10 mg/kg bw, s.c.), the neurons of the caudal trigeminal nucleus (TNC) are activated and the expression of neuronal NO synthase (nNOS) in the same area is increased suggesting a self-amplifying process in the trigeminal system, which seems to be crucial in migraine pathogenesis. Kynurenic acid (KYNA) and its analogues may exert modulatory effects in many neuropathological conditions, probably via N-methyl-D-aspartate (NMDA) antagonism. Since NMDA receptors play a crucial role in trigeminal pain processing, the aim of our experiments was to compare the effects of L-kynurenine (L-KYN) combined with probenecid (PROB) or with 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1H-quinolin-4-one hydrochloride alone, a newly synthetized KYNA derivative, on the NTG-induced nNOS expression in the rat TNC. Pretreatment with L-KYN (300 mg/kg bw, i.p.) together with PROB (200 mg/kg bw, i.p.) and KYNA derivative (300 mg/kg bw, i.p.) attenuated the NTG-induced nNOS expression in the rat TNC. Our data suggest that the stimulating effect of NTG, and thus of NO, on the expression of nNOS might be modulated by increasing the KYNA level in the brain, probably through the NMDA receptors. These data could help promote a better understanding of the pathogenesis of headaches and the action of antimigraine drugs.

Neuroprotective effects of probenecid in a transgenic animal model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominantly inherited disorder, caused by an expanded polyglutamine region of a protein called huntingtin. The excitotoxicity, oxidative damage and altered membrane transport may have an important role in the pathogenesis of HD. Probenecid is a non-selective inhibitor of multidrug resistance-associated proteins, but it also inhibits organic anion transporters. In this study, we examined the effects of probenecid on the survival, behaviour and immunohistochemical changes in the N171-82Q transgenic mouse model of HD. After probenecid administration, the duration of survival improved by 35%. The motor activity was significantly ameliorated as compared with the control transgenic group. Probenecid treatment significantly reduced the neuronal loss and the number of neuronal intranuclear aggregates. These results suggest that probenecid may exert a neuroprotective effect by increasing the membrane transport of protective compounds, and/or inhibiting the toxic compounds.

The role of kynurenines in disorders of the central nervous system: possibilities for neuroprotection.

The metabolism of tryptophan mostly proceeds through the kynurenine pathway. The biochemical reaction includes both an agonist (quinolinic acid) at the N-methyl-d-aspartate receptor and an antagonist (kynurenic acid). Besides the N-methyl-d-aspartate antagonism, an important feature of kynurenic acid is the blockade of the alpha7-nicotinic acetylcholine receptor and its influence on the alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptor. Kynurenic acid has proven to be neuroprotective in several experimental settings. On the other hand, quinolinic acid is a potent neurotoxin with an additional and marked free radical-producing property. In consequence of these various receptor activities, the possible roles of these substances in various neurological disorders have been proposed. Moreover, the possibility of influencing the kynurenine pathway to reduce quinolinic acid and increase the level of kynurenic acid in the brain offers a new target for drug action designed to change the balance, decreasing excitotoxins and enhancing neuroprotectants. This review surveys both the early and the current research in this field, focusing on the possible therapeutic effects of kynurenines.

Selective inhibition of cyclooxygenase-2 attenuates nitroglycerin-induced calmodulin-dependent protein kinase II alpha in rat trigeminal nucleus caudalis.

The nitric oxide donor, nitroglycerin (NTG) can trigger a migraine attack, after a delay of several hours in migraineurs, but not in healthy people. This long delay does not favor a pure vasodilatatory action. In rats, subcutaneous administration of NTG (10mg/kg) significantly and selectively increases the number of calmodulin-dependent protein kinase II alpha (CamKIIalpha)-immunoreactive neurons in the trigeminal caudal nucleus (TNC) after 4h. The aim of our study was to determine if any isoforms of the cyclooxygenase (COX) enzyme might have a role in the NTG-induced increase of CamKIIalpha expression. In our experiments, we demonstrated that pretreatment with NS398, the selective COX-2 inhibitor attenuated the NTG-induced CamKIIalpha expression in the TNC at doses of 3 and 5mg/kg. In contrast, SC560, a selective COX-1 inhibitor failed to modulate this phenomenon in any of the dosages used (1, 5 and 10mg/kg). These findings suggest that COX-2, but not COX-1 derived metabolites are important factors in the NTG-induced CamKIIalpha expression. Thus this isoform may play a significant role in the induction of migraine. These data could help in the better understanding of the pathogenesis of headaches and the action of antimigraine drugs.

Effects of valproate on the dopaminergic system in mice.

Valproate is a widely used antiepileptic drug with a known tremor-inducing effect. In this study, we tested the behavioral changes induced by valproate and its effect on the dopaminergic cells in the substantia nigra of mice. This treatment led to a spontaneous locomotor activity, while the explorative behavior was unaltered. The number of dopaminergic cells in the substantia nigra was reduced after valproate treatment. These data are indicative of dopaminergic toxicity, but further studies are needed to demonstrate the exact mechanism.

Cox-2 inhibitor attenuates NO-induced nNOS in rat caudal trigeminal nucleus.

OBJECTIVE: The aim of the present study was to determine which isoform of the cyclooxygenase (COX) enzyme plays a role in the neuronal nitric oxide synthase (nNOS) activation caused by nitroglycerin (NTG), in the most caudal part of the trigeminal caudal nucleus (TNC) of the rat. BACKGROUND: Nitric oxide donor, NTG, can trigger migraine attack in migraineurs, but not in healthy persons. In rats, subcutaneous administration of NTG (10 mg/kg) increases significantly the number of nNOS-immunoreactive neurons in the TNC after 4 hours, which could be attenuated by acetyl-salicylate (Aspirin), a nonselective COX-inhibitor. METHODS: SPRD rats were divided into 3 groups: (1) control group (no drug administration), (2) NS398 (selective COX-2 inhibitor) administration (1, 3, or 5 mg/kg), and (3) SC560 (selective COX-1 inhibitor) administration (1, 5, or 10 mg/kg). Thirty minutes after drug administration, the animals received NTG (10 mg/kg) or placebo injection. Four hours later the animals were transcardially perfused and the cervical part of the TNC was removed for immunohistochemistry. Results.-The selective COX-2 inhibitor NS398 in contrast to the selective COX-1 inhibitor SC560 attenuates the NTG-induced nNOS expression dose-dependently. CONCLUSION: These findings suggest that metabolites deriving from COX-2 (but not COX-1) may be the most important factors in the NTG-induced nNOS expression. These data could help to better understand the pathogenesis of headaches and the action of antimigraine drugs.