Synthesis, characterization, and biological study of new synthetic opioid hemorphin-4 peptides containing sterically restricted nonnatural amino acids.

Some new hemorphin-4 analogs with structures of Xxx-Pro-Trp-Thr-NH2 and Tyr-Yyy-Trp-Thr-NH2, where Xxx is 2-amino-3-(4-hydroxy-2,6-dimethylphenyl)propanoic acid or 2-amino-3-(4-dibenzylamino-2,6-dimethylphenyl)propanoic acid, and Yyy is (2S,4S)-4-amino-pyrrolidine-2-carboxylic acid, were synthesized and characterized by electrochemical and spectral analyses. In vivo anticonvulsant and antinociceptive activities of peptide derivatives were studied after intracerebroventricular injection in mice. The therapeutic effects of the modified peptides on seizures and pain in mice were evaluated to provide valuable insights into the potential applications of the novel compounds. Electrochemical characterization showed that the compounds behave as weak protolytes and that they are in a soluble, stable molecular form at physiological pH values. The antioxidant activity of the peptides was evaluated with voltammetric analyses, which were confirmed by applying the 2,2-Diphenyl-1-picrylhydrazyl method. The compounds showed satisfactory results regarding their structural stability, reaching the desired centers for the manifestation of biological activity without hydrolysis processes at 37°C and physiological pH. Dm-H4 and H4-P1 exhibited 100% and 83% potency to suppress the psychomotor seizures in the 6-Hz test compared to 67% activity of H4. Notably, only the H4-P1 had efficacy in blocking the tonic component in the maximal electroshock test with a potency comparable to H4. All investigated peptides containing unnatural conformationally restricted amino acids showed antinociceptive effects. The analogs Db-H4 and H4-P1 showed the most pronounced and long-lasting effect in both experimental models of pain induced by thermal and chemical stimuli. Dm-H4 produced a dose-dependent thermal antinociception and H4-P2 inhibited only formalin-induced pain behavior.

Synthesis, molecular docking, electrochemical and fluorimetric analysis of new caffeic and cinnamic acid-conjugated hemorphin derivatives designed as potential anticonvulsant and antinociceptive agents.

Based on the pharmacophore model of opioid receptors, our team recently synthesized a series of short-chain hemorphin peptide analogs containing non-natural amino acids. They demonstrated anticonvulsant and antinociceptive activity with low neurotoxicity. In the present study, a series of novel bioconjugates of N-modified hemorphin analogs containing second pharmacophore cinnamic acids (CA) or caffeic (KA) were synthesized by a traditional solid-phase Fmoc chemistry method for peptide synthesis. Electrochemical and fluorimetric analysis, in vivo anticonvulsant and antinociceptive activity in mice were conducted on the compounds. The three CA acid- (H4-CA, H5-CA, and H7-CA) and three KA acid- (H4-KA, H5-KA, and H7-KA) conjugated hemorphin derivatives exhibited potency at the highest doses of 2 µg/5 µl, administered by intracerebroventricular (icv) mode, against seizure spread in the maximal electroshock test (MES) in mice. The KA-conjugated H5-KA derivate, at the lowest dose, was the only compound that suppressed clonic seizures in the subcutaneous pentylenetetrazol (scPTZ) test. Except for the H5-CA, all tested CA acid- and KA acid-conjugated peptide derivates had the potency to increase the latency for clonic seizures in a dose-dependent mode. The activity against the psychomotor seizures in the 6-Hz test was detected only for the H4-CA (0.5 µg) and H4-KA (0.5 µg and 1 µg), respectively. All investigated peptides showed a more pronounced antinociceptive effect in the "intraplantar formalin" test compared to the "hot plate" test. Shorter chain analogs showed a better antinociceptive profile against tonic pain. The data suggest a DOR and KOR-mediated mechanism of action. According to the docking analysis, H7-CA showed a different antinociceptive profile than other investigated peptides. The novel peptide derivates did not exhibit neurotoxicity in the rotarod test. Our findings suggest that conjugated CA and KA morphine peptides can be used to develop novel morphine-related analogs with anticonvulsant and antinociceptive activity.

Behavioral Effects and Analgesic Profile of Hemoglobin-Derived Valorphin and Its Synthetic Analog in Rodents.

Valorphin (V1) is a naturally occurring peptide derived from hemoglobin that has been found to have an affinity for opioid receptors and exhibits antinociceptive and anticonvulsant activity. Some of its synthetic analogs containing an aminophosphonate moiety show structure-dependent potent antinociceptive effects. This study aimed to reveal a detailed picture of the antinociceptive mechanisms and behavioral effects of V1 and its recently synthesized phosphopeptide analog V2p in rodents using a range of methods. The studied peptides significantly reduced acute (mean V1-9.0, V2p-5.8 vs. controls-54.1 s) and inflammatory (mean V1-57.9 and V2p-53.3 vs. controls-107.6 s) nociceptive pain in the formalin test, as well as carrageenan-induced hyperalgesia (mean V1-184.7 and V2p-107.3 vs. controls-61.8 g) in the paw pressure test. These effects are mediated by activation of opioid receptors with a predominance of kappa in V1 antinociception and by delta, kappa, and mu receptors in V2p-induced antinociception. Both peptides did not change the levels of TNF-alpha and IL-1-beta in blood serum. V1 induces depression-like behavior, and V2p shows a tendency toward anxiolysis and short-term impairment of motor coordination without affecting exploratory behavior. The results characterize valorphin and its derivative as promising analgesics that exert their effects both centrally and peripherally, without causing severe behavioral changes in experimental animals. These encouraging data are a foundation for future studies focusing on the effects of hemorphins after long-term treatment.

Synthesis, characterization and evaluation of anti-hyperalgesia, anticonvulsant and antioxidant activity of novel VV-hemorphin-5 analogs.

Two series of new VV-hemorphin-5 analogs with structures Val-Val-Tyr-Xxx-Trp-Thr-Gln-NH2 and Adam-Val-Val-Tyr-Xxx-Trp-Thr-Gln-NH2 , where Xxx is Ac5c (1-aminocyclopentane-1-carboxylic acid), Ac6c (1-aminocyclohexane-1-carboxylic acid), Ac7c (1-aminocycloheptane-1-carboxylic acid), and Adam is the low-molecular-weight lipophilic adamantyl building block, were synthesized, characterized electrochemically and evaluated for antioxidant, anti-hyperalgesia, and anticonvulsant activity. The design of the compounds followed the strategy to improve the propensity for aqueous solubility and/or to increase their affinity for the target receptor or enzyme. The partition coefficient value shows that the peptide scaffold goes from hydrophilic to lipophilic with the increasing size of the cycloalkane ring and even more with the introduction of the adamantane. The peptides C5-V and C7-V were the only analogs that provoked an immediate antinociceptive effect changing the mechanical pain threshold. The six new peptide analogs produced a significant and long-lasting carrageenan model of inflammatory pain in rats. While the adamantane hemorphin analog Ad7-V was the only compound with the potency to suppress psychomotor seizures in the 6-Hz test, the C6-V and Ad6-V exhibited protective activity against the seizure spread in the maximal electroshock seizure test in mice. The active analogs did not show neurotoxicity or sedative effects. Our results revealed a structure-related specific activity of a newly designed hemorphin analog that could be used as a template for future modification and preparation of compounds with potential analgesic and anticonvulsant activity.

New N- and C-modified RGD-hemorphins as potential biomedical application on Ti-surface materials: synthesis, characterization and antinociceptive activity.

This manuscript presented the synthesis and characterization of two new N- and C-modified analogues of VV-hemorphin-7 containing RGD (Arg-Gly-Asp) residues as potential nociceptive agents and bioactive materials. It has been shown that the addition of one or two RGD sequences to natural VV-hemorphin-7 increases its effect on acute nociception, but the reduction of the inflammatory phase depends on the concentration of the peptide. The structure-property relationship of the new peptide derivatives was highlighted by electrochemical and FT-IR methods of analysis. Because of the proven bone-structural bonds of hydroxyapatite, the simultaneous deposition of peptide/hydroxyapatite on the surface of a titanium surface was investigated. The deposition was performed in a medium of gelatin solution containing dissolved amounts of peptide and hydroxyapatite using ultrasound. SEM-EDS analyzes confirmed the presence of a layer of the studied system.

Effects of the antinociceptive dipeptide L-tyrosine-L-arginine (kyotorphin) on the motivation, anxiety, and memory in rats.

INTRODUCTION: The endogenous dipeptide L-tyrosine-L-arginine (kyotorphin, KTP) is found in brain structures related to the processing of information for nociception, the control of emotions, and memory formation. Besides the antinociceptive effect of KTP, it has a mild protective activity against the deleterious influence of the brain hypoperfusion and streptozotocin on the behavior and memory. AIM: We aimed to study the effects of the intracerebroventricular injection of effective antinociceptive doses of KTP on the motivational behavior, memory, and blood and hippocampal levels of the carbonylated proteins in healthy male adult Wistar rats. MATERIALS AND METHODS: We used a paw-pressure test for assessment of acute nociception, an open field test for assessment of exploration and habituation to a new environment, elevated plus maze test for the evaluation of anxiety-like behavior, and novel object recognition test for working memory. Carbonylated protein assay was used for the assessment of the oxidative impairment of the proteins. The results were analyzed by ANOVA. RESULTS: The present data showed that all single doses of KTP exerted an antinociceptive effect, but this effect was not observed after chronic administration. Only the highest dose of 100 µg was able to induce anxiolytic and motor inhibiting effects. None of the doses used showed effects on the recognition memory or the level of the carbonylated protein. CONCLUSION: Our results showed that KTP exerted its antinociceptive effect without affecting negatively the blood and brain carbonylated protein or basic behavioral parameters related to the exploration, motivation, and memory formation in healthy rats.

GC-MS analysis of Amaryllidaceae and Sceletium-type alkaloids in bioactive fractions from Narcissus cv. Hawera.

RATIONALE: Narcissus cv. Hawera has been found to biosynthesize some Sceletium-type alkaloids with antidepressant and anxiolytic activities. This ornamental plant has been poorly studied as a source of bioactive alkaloids including some contraversive reports on in vitro and intact plants. In this study, a detailed GC-MS characterization of its alkaloid fractions is presented. METHODS: GC-MS was used for the identification of compounds in the alkaloid fractions. Both underivatized and silylated samples were analyzed simultaneously. Elevated plus maze and tail suspension tests were used to assay the anxiolytic and antidepressant activities. Ellman's and MTT-dye reduction assays were used to evaluate the acetylcholinesterase (AChE) inhibitory and cytotoxicity activities, respectively. RESULTS: Of the 29 alkaloids, 13 of Sceletium-type were detected. Two new alkaloids were identified as 2-oxo-mesembrine and 2-oxo-epi-mesembrenol. Lycorine was found as a major compound (43.5%) in the crude silylated methanol extract. After the elimination of lycorine by pre-crystallization, the major alkaloids were 40.8% 6-epi-mesembranol, 16.2% 6-epi-mesembrenol, and 13.8% sanguinine. This fraction showed anxiolytic and antidepressant-like activities as well as potent AChE inhibitory and antineoplastic activities. CONCLUSIONS: Silylation of the alkaloid fractions from Narcissus cv. Hawera provides better separation, structural information, and improved sensitivity for compounds with two and more hydroxyl groups. The lycorine-free alkaloid fraction shows a great potential for further pharmacological studies.

Antinociceptive Effects of VV-Hemorphin-5 Peptide Analogues Containing Amino phosphonate Moiety in Mouse Formalin Model of Pain.

BACKGROUND: Hemorphins are endogenous hemoglobin-derived peptides that belong to the family of "atypical" opioid peptides with both affinities to opioid receptors and ability to release other endogenous opioid peptides. OBJECTIVE: In the present study, peptide analogues of Valorphin (VV-hemorphin-5) containing amino phosphonate moiety synthesized by solid-phase peptide synthesis (Fmoc-strategy) were investigated for their potential antinociceptive activities and compared to the reference VV-H in formalin- induced model of acute and inflammatory pain in mice. METHODS: The hemorphin analogues were prepared by replacement of the one and/or two N-terminal Val in VV-hemorphin5 (VV-H) with ((dimethoxy phosphoryl) methyl)-L-valine and ((dimethoxy phosphoryl) methyl)-L-leucine to obtain the compounds pVV-H, pL-H, and pLV-H. Aiming to additionally prove the importance of amino acid valine, we introduced the ((dimethoxy phosphoryl) methyl)-L-leucine to the N-side of VV-hemorphin-5 (pLVV-H). The experiments were carried out on adult male ICR mice. All peptides were administered intracerebroventricularly at three doses (50, 25 and 12,5 µg/mouse). We have studied the effects of the peptides on acute (1st phase) and inflammatory (2nd phase) pain reaction using un experimental model with intraplantar formalin injection. RESULTS: VV-H showed a significant antinociceptive effect both in the acute and inflammatory phases of the test. Although Valorphin hexa-, hepta-, and octapeptide analogs demonstrated a significant antinociceptive effect, they showed substantial differences considering their effective dose and the phase of the test as compared to the Valorphin. DISCUSSION: Data showed that modified heptapeptides pVV-H and pLV-H exerted the same or better antinociception in acute and inflammatory pain, in comparison to the reference peptide, while pL-H and pLVV-H analogues were less effective. CONCLUSION: Our study contributes to the elucidation of the role of Valine and the number of amino acid residues in the structure of hemorphin peptide analogs in their effectiveness in suppressing both acute and inflammatory experimental pain.

Protective effects of losartan on some type 2 diabetes mellitus-induced complications in Wistar and spontaneously hypertensive rats.

Diabetes mellitus type 2 (T2DM) is characterized by resistance of insulin receptors and/or inadequate insulin secretion resulting in metabolic and structural complications including vascular diseases, arterial hypertension and different behavioral alterations. We aimed to study the effects of the antihypertensive angiotensin AT1 receptor antagonist losartan on the T2DM-induced changes of exploratory behavior, anxiety, nociception and short term memory in normotensive Wistar and spontaneously hypertensive rats (SHRs). The experimental model of T2DM induced by a combination of high fat diet and streptozotocin, decreased exploratory activity and increased the level of carbonylated proteins in selected brain structures in both strains; as well it increased corticosterone level, pain threshold, anxiety-like behavior, and decline short term memory only in SHRs. Losartan treatment alleviated some of the T2DM- induced metabolic complications, abolished the T2DM-induced hypo activity, and normalized the corticosterone level, carbonylated proteins in brain, nociception and memory. Losartan did not exert effect on the anxiety behavior in both strains. We showed that T2DM exerted more pronounced negative effects on the rats with comorbid hypertension as compared to normotensive rats. Overall effects on the studied behavioral parameters are related to decreased exploration of the new environment, increased anxiety-like behavior, and decline in short-term memory. The systemic sub-chronic treatment with an angiotensin AT1 receptor antagonist losartan ameliorated most of these complications.

Moderate protective effect of Kyotorphin against the late consequences of intracerebroventricular streptozotocin model of Alzheimer's disease.

The established decrease in the level of endogenous kyotorphin (KTP) into the cerebrospinal fluid of patients with an advanced stage of Alzheimer's disease (AD) and the found neuroprotective activity of KTP suggested its participation in the pathophysiology of the disease. We aimed to study the effects of subchronic intracerebroventricular (ICV) treatment (14 days) with KTP on the behavioral, biochemical and histological changes in rats with streptozotocin (STZ-ICV)-induced model of sporadic AD (sAD). Three months after the administration of STZ-ICV, rats developed increased locomotor activity, decreased level of anxiety, impaired spatial and working memory. Histological data from the STZ-ICV group demonstrated decreased number of neurons in the CA1 and CA3 subfields of the hippocampus. The STZ-ICV group was characterized with a decrease of total protein content in the hippocampus and the prefrontal cortex as well as increased levels of the carbonylated proteins in the hippocampus. KTP treatment of STZ-ICV rats normalized anxiety level and regained object recognition memory. KTP abolished the protein loss in prefrontal cortex and decrease the neuronal loss in the CA3 subfield of the hippocampus. STZ-ICV rats, treated with KTP, did not show significant changes in the levels of the carbonylated proteins in specific brain structures or in motor activity and spatial memory compared to the saline-treated STZ-ICV group. Our data show a moderate and selective protective effect of a subchronic ICV administration of the dipeptide KTP on the pathological changes induced by an experimental model of sAD in rats.

Synthesis, characterization and nociceptive screening of new VV-hemorphin-5 analogues.

In the present study, some new analogues of VV-hemorphin-5, modified at position 1 and 7 by the non-proteinogenic and/or natural amino acids followed the structures Xxx-Val-Val-Tyr-Pro-Trp-Thr-Gln-NH2 and Val-Val-Tyr-Pro-Trp-Thr-Yyy-NH2, where Xxx is Ile or Aib and Yyy is Lys/Orn/Dap/Dab were synthesized to investigate their potential antinociceptive activities. We report also the redox potentials and the acid/base properties as pKa values of these peptide analogues which were compared toward electrochemical behaviour of tryptophan containing peptides. All analogues showed a short lasting initial antinociceptive effect, however H2 hemorphin analogue is characterized with prolong and strong antinociceptive effect, while the other peptide analogues exerted more variable effects on the visceral nociception depending on the dose or time after the intracerebral injection.

Long-Term Treatment with Losartan Attenuates Seizure Activity and Neuronal Damage Without Affecting Behavioral Changes in a Model of Co-morbid Hypertension and Epilepsy.

Over the last 10 years, accumulated experimental and clinical evidence has supported the idea that AT1 receptor subtype is involved in epilepsy. Recently, we have shown that the selective AT1 receptor antagonist losartan attenuates epileptogenesis and exerts neuroprotection in the CA1 area of the hippocampus in epileptic Wistar rats. This study aimed to verify the efficacy of long-term treatment with losartan (10 mg/kg) after kainate-induced status epilepticus (SE) on seizure activity, behavioral and biochemical changes, and neuronal damage in a model of co-morbid hypertension and epilepsy. Spontaneous seizures were video- and EEG-monitored in spontaneously hypertensive rats (SHRs) for a 16-week period after SE. The behavior was analyzed by open field, elevated plus maze, sugar preference test, and forced swim test. The levels of serotonin in the hippocampus and neuronal loss were estimated by HPLC and hematoxylin and eosin staining, respectively. The AT1 receptor antagonism delayed the onset of seizures and alleviated their frequency and duration during and after discontinuation of treatment. Losartan showed neuroprotection mostly in the CA3 area of the hippocampus and the septo-temporal hilus of the dentate gyrus in SHRs. However, the AT1 receptor antagonist did not exert a substantial influence on concomitant with epilepsy behavioral changes and decreased 5-HT levels in the hippocampus. Our results suggest that the antihypertensive therapy with an AT1 receptor blocker might be effective against seizure activity and neuronal damage in a co-morbid hypertension and epilepsy.

Long-term intracerebroventricular infusion of angiotensin II after kainate-induced status epilepticus: Effects on epileptogenesis, brain damage, and diurnal behavioral changes.

Our previous studies revealed that Angiotensin (Ang) II has anticonvulsant effects in acute seizure models. However, data on its role in experimental models of epilepsy are missing. In the present study, we tested whether posttreatment with Ang II after kainate (KA)-induced status epilepticus (SE) can affect epileptogenesis, concomitant behavioral changes, and brain damage. The Wistar rats were intracerebroventricularly infused via osmotic mini-pumps with Ang II (1.52µg/µl/day for 28days) after SE. Spontaneous motor seizures (SMS) were video-recorded for up to three months. Locomotor activity, anxiety, and depression-like behavior were evaluated during the last week of drug infusion, while spatial memory was assessed during the 3rd month after SE. Angiotensin II decreased the latency for onset of the first SMS and increased the frequency of SMS two months after SE. The continuous peptide infusion exacerbated the KA-induced hyperactivity and caused depression-like behavior. The reduced anxiety of KA-treated rats was alleviated by Ang II exposure. The KA-induced deficit in the hippocampal-dependent spatial memory was not influenced by Ang II. However, Ang II partially prevented the neuronal damage in the hippocampus, specifically in the CA1 area. The role of AT1 and AT2 receptor activation in the effects of the octapeptide is discussed.

Antiepileptogenic and neuroprotective effects of losartan in kainate model of temporal lobe epilepsy.

Recently, we have shown that the blockade of AT1 receptor might be useful as an adjuvant treatment strategy for the prevention of oxidative stress and neurotoxicity caused by status epilepticus (SE) in rats. The purpose of the present study was to further assess the efficacy of long-term treatment with losartan (10mg/kg), the selective AT1 receptor antagonist, during kainate (KA)-induced epileptogenesis in Wistar rats. Losartan treatment started after onset of SE and continued for 4weeks. The rats were video- and EEG-recorded for 3months. Locomotor activity, anxiety and depressive-like behavior were evaluated 9weeks after SE, when all rats had developed chronic epileptic state. Neuronal damage in hippocampus was analyzed by hematoxylin while serotonin (5-HT) levels in hippocampus by HPLC. AT1 receptor antagonism increased the latent seizure-free period and decreased the frequency of spontaneous motor seizures. Losartan positively affected epilepsy-provoked behavioral changes, including impulsivity, low anxiety level and depression in a phase-dependent manner and restored the changes in diurnal fluctuation of motor activity. Losartan exerted neuroprotection selectively in the CA1 area of the hippocampus in the KA-treated rats and lowered the 5-HT levels both in normal and abnormal conditions. Our findings suggest that the AT1 receptor antagonist exerts disease-modifying effects during KA-induced epileptogenesis and neuronal damage in CA1 hippocampal area, attenuated some of the behavioral changes and restored diurnal variability in locomotor activity.

Treatment with melatonin after status epilepticus attenuates seizure activity and neuronal damage but does not prevent the disturbance in diurnal rhythms and behavioral alterations in spontaneously hypertensive rats in kainate model of temporal lobe epilepsy.

Melatonin is involved in the control of circadian and seasonal rhythmicity, possesses potent antioxidant activity, and exerts a neuroprotective and anticonvulsant effect. Spontaneously hypertensive rats (SHRs) are widely accepted as an experimental model of essential hypertension with hyperactivity, deficient sustained attention, and alterations in circadian autonomic profiles. The purpose of the present study was to determine whether melatonin treatment during epileptogenesis can prevent the deleterious consequences of status epilepticus (SE) in SHRs in the kainate (KA) model of temporal lobe of epilepsy (TLE). Spontaneous recurrent seizures (SRSs) were EEG- and video-recorded during and after the treatment protocol. Melatonin (10mg/kg diluted in drinking water, 8weeks) increased the seizure-latent period, decreased the frequency of SRSs, and attenuated the circadian rhythm of seizure activity in SHRs. However, melatonin was unable to affect the disturbed diurnal rhythms and behavioral changes associated with epilepsy, including the decreased anxiety level, depression, and impaired spatial memory. Melatonin reduced neuronal damage specifically in the CA1 area of the hippocampus and piriform cortex and decreased hippocampal serotonin (5-HT) levels both in control and epileptic SHRs. Although long-term melatonin treatment after SE shows a potential to attenuate seizure activity and neuronal loss, it is unable to restore epilepsy-associated behavioral abnormalities in SHRs.

Strain-dependent effects of sub-chronically infused losartan against kainic acid-induced seizures, oxidative stress, and heat shock protein 72 expression.

We studied the involvement of angiotensin (Ang) II AT1 receptors in the pathophysiology of kainate (KA)-induced neurotoxicity, focusing on the regulation of the oxidative stress state and expression of HSP 72 in the frontal cortex and hippocampus in two strains, spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. The KA injection was executed after the rats were infused subcutaneously via osmotic mini-pumps with losartan (10 mg/kg day) for 14 days. Losartan delayed the onset of KA-induced seizures in SHRs but not in Wistar rats without affecting the seizure intensity score. This selective AT1 receptor antagonist decreased the lipid peroxidation only in naive SHRs. However, it attenuated the KA-induced increase in lipid peroxidation in both SHRs and Wistar rats. The adaptive enhancement of cytosolic superoxide dismutase (SOD) activity in KA-treated SHRs was recovered to control level after sub-chronic losartan infusion while no change in mitochondrial SOD activity was detected in the two strains. Both losartan and KA produced a higher expression of HSP 72 in the hippocampus of the two strains compared to naive rats infused with vehicle. Taken together, our findings demonstrate that the efficacy of a sub-chronic systemic losartan infusion in preventing the KA-induced seizure activity and neurotoxicity is more pronounced in SHRs, considered as a model of essential hypertension, than in normotenisve Wistar rats. The results suggest that the blockade of AT1 receptors, commonly used as a strategy for prevention of high blood pressure, may be useful as an adjunctive treatment in status epilepticus to reduce oxidative stress and neurotoxicity.

Strain-dependent effects of long-term treatment with melatonin on kainic acid-induced status epilepticus, oxidative stress and the expression of heat shock proteins.

Oxidative stress is implicated in the pathogenesis of both hypertension and epileptogenesis, therefore it could be used as a tool for studying co-morbidity of hypertension and epilepsy. Clinical data suggest that melatonin is a potent antioxidant that is effective in the adjunctive therapy of hypertension and neurodegenerative diseases. The present study aimed to explore and compare the efficacy of chronic pretreatment with melatonin infused via subcutaneous osmotic mini-pumps for 14 days (10 mg/kg per day) on kainic acid (KA)-induced status epilepticus, oxidative stress and expression of heat shock protein (HSP) 72 in spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. SHRs showed higher lipid peroxidation (LP) in the frontal cortex and hippocampus and decreased cytosolic superoxide dismutase (SOD/CuZn) production in the frontal cortex compared to Wistar rats. Status epilepticus (SE) induced by KA (12 mg/kg, i.p.) was accompanied by increased LP and expression of HSP 72 in the hippocampus of the two strains and increased SOD/CuZn production in the frontal cortex of SHRs. Melatonin failed to suppress seizure incidence and intensity though the latency for seizure onset was significantly increased in SHRs. Melatonin attenuated the KA-induced increase in the level of LP in the hippocampus both in SHRs and Wistar rats. However, an increased activity in SOD/CuZn and mitochondrial SOD Mn as well as reduced expression of HSP 72 in the hippocampus was observed only in Wistar rats pretreated with melatonin. Taken together, the observed strain differences in the efficacy of chronic melatonin exposure before SE suggest a lack of a direct link between the seizure activity and the markers of oxidative stress and neurotoxicity.

Prophylactic treatment with melatonin after status epilepticus: effects on epileptogenesis, neuronal damage, and behavioral changes in a kainate model of temporal lobe epilepsy.

Melatonin is a potent antioxidant which showed anticonvulsant activities both in experimental and clinical studies. In the present study, we examined the effect of melatonin treatment (10mg/kg/day, diluted in drinking water, 8 weeks) during epileptogenesis on the consequences of a kainate (KA)-induced status epilepticus (SE) in rats. Melatonin increased the latency in the appearance of spontaneous recurrent seizures (SRSs) and decreased their frequency only during the treatment period. The behavioral alterations associated with hyperactivity, depression-like behavior during the light phase, and deficits in hippocampus-dependent working memory were positively affected by melatonin treatment in rats with epilepsy. Melatonin reduced the neuronal damage in the CA1 area of the hippocampus and piriform cortex and recovered the decrease of hippocampal serotonin (5-HT) level in rats with epilepsy. Taken together, long-term melatonin treatment after SE was unable to suppress the development of epileptogenesis. However, it showed a potential in reducing some of the deleterious alterations that develop during the chronic epileptic state in a diurnal phase-dependent mode.

Effect of chronic treatment with angiotensin receptor ligands on water-salt balance in Wistar and spontaneously hypertensive rats.

UNLABELLED: The renin-angiotensin system plays a crucial role in the regulation of cardiovascular function and maintenance of water-electrolyte balance. The two major receptor types of the system, AT1 and AT2, have different, often opposite effects on these functions. AIM: To elucidate the impact of long-term treatment with selective angiotensin receptor antagonists and an agonist on water-salt balance in normotensive Wistar and spontaneously hypertensive rats (SHRs). MATERIALS AND METHODS: 12-week-old male Wistar rats and SHRs were individually housed in metabolic cages and 24-h food and water intake and urine and electrolyte excretion were measured. Urinary sodium (UNa), potassium (UK) and chlorine (UCl) were determined by a flame photometer. Losartan, a selective AT1 receptor antagonist, was administered in the Wistar rats and SHRs at a dose of 10 mg/kg/day subcutaneously (sc). Wistar rats were also given the AT2 receptor antagonist, PD123319, subcutaneously at a dose of 10 mg/kg/ day. CGP 42112A, an AT2 receptor agonist, was administered intracerebroventricularly in Wistar rats at a dose of 12 microg/rat/day. The drugs were infused continuously for 14 days through osmotic minipumps. RESULTS: Losartan selectively increased sodium excretion in both rat strains and decreased weight gain in SHRs. PD123319 increased potassium excretion and decreased weight gain in Wistar rats. CGP 42112A increased food and water intake, urine output and UNa+ and UK+ excretion and decreased weight gain in normotensive Wistar rats. CONCLUSIONS: Chronic treatment with selective angiotensin receptor ligands modifies water-salt balance in rats through changes both in renal excretory function and ingestive behaviors.

Effect of long-term caffeine administration on depressive-like behavior in rats exposed to chronic unpredictable stress.

Chronic unpredictable stress (CUS) was used to study the effects of a long-term treatment with either caffeine (8 mg/kg, orally) or desipramine (DMI) (10 mg/kg, intraperitoneally) in Wistar rats. The CUS procedure was applied for 6 weeks. Animals underwent a 2-week drug-free CUS procedure. Drugs were administered for 4 weeks alongside the stress and both drug and stress were continued throughout the behavioral testing period. CUS-exposed rats showed depressive-like behavior with reduced weight gain, reduced consumption of sucrose solution, increased immobility in the forced swimming test, and hypolocomotion in an open field. For the open field and elevated plus maze, calculation of an anxiety index confirmed that CUS increased anxiety, which was accompanied by an increase in the core temperature. DMI counteracted these physical and behavioral changes. Caffeine caused similar effects to DMI on weight gain, motor activity, anxiety level, and core temperature. In CUS-exposed rats, caffeine showed antidepressant and anxiolytic activity, accompanied by increased hippocampal dopamine and serotonin levels. However, no significant change in weight gain or core temperature was observed after caffeine treatment in CUS-exposed rats. These results suggest that, similar to the antidepressant DMI, long-term caffeine exposure exerts an antidepressant and anxiolytic effect in the CUS model. The involvement of the dopaminergic and serotonergic systems is discussed.