Seizure duration following sarin exposure affects neuro-inflammatory markers in the rat brain.

The current study was aimed to characterize for the first time the alterations in the characteristic neuro-inflammatory markers triggered by sarin exposure in the rat's brain, and to investigate its dependency on seizure duration. Centrally mediated seizures are a common consequence of exposure to organophosphates (OP) despite conventional treatment with atropine and an oxime. In the present study midazolam, was used to control duration and intensity of seizures. The levels of the pro-inflammatory cytokine peptides IL-1beta, IL-6, TNF-alpha and prostaglandin E2 (PGE2) were monitored at various times after sarin exposure in the hippocampus and cortex of rats treated with midazolam following 5 or 30 min of seizure activity. Biochemical evaluation of brain tissues revealed a significant increase in the level of the pro-inflammatory peptides starting at 2 h and peaking at 2-24 h following sarin. Hippocampal values of IL1-beta increased from 1.2+/-0.1 pg/mg tissue (control), to 2.4+/-0.3 at 2 h (5 min seizure) and to 9.3+/-2.5 at 8h (30 min seizure). PGE2 level in the hippocampus increased up to 24 h following exposure (from 56+/-3 to 175+/-26 and 277+/-28 pg/mg tissue) following 5 and 30 min of seizure activity respectively. Thus, unlike limitation of seizures to 5 min by midazolam, delayed treatment (30 min) resulted in prolonged seizures and pronounced increase in cytokines and PGE2. In addition, a second increase in inflammatory markers was observed 30 days following sarin exposure only in rats treated following 30 min of seizure activity. Histological evaluation of the rat brain, conducted in this study, revealed lack of damage in the hippocampus and piriform cortex with minor lateral ventricles enlargement in few animals following 5 min of sarin-induced seizure activity. In contrast, marked histological damage to the brain was demonstrated following 30 min of seizure activity, consisting severe damage to the hippocampus, piriform cortex and some thalamic nuclei. In summary, a novel characterization of the prolonged central neuro-inflammatory process that accompanies sarin exposure is presented. The timing of the anticonvulsive treatment was shown to be crucial in modulation of the neuro-inflammatory response, and may implicate the consequent long-term brain damage.

Oxinium modular femoral heads do not reduce polyethylene wear in cemented total hip arthroplasty at five years: a randomised trial of 120 hips using radiostereometric analysis.

We report the five-year outcome of a randomised controlled trial which used radiostereometric analysis (RSA) to assess the influence of surface oxidised zirconium (OxZr, Oxinium) on polyethylene wear in vivo. A total of 120 patients, 85 women and 35 men with a mean age of 70 years (59 to 80) who were scheduled for primary cemented total hip arthroplasty were randomly allocated to four study groups. Patients were blinded to their group assignment and received either a conventional polyethylene (CPE) or a highly cross-linked (HXL) acetabular component of identical design. On the femoral side patients received a 28 mm head made of either cobalt-chromium (CoCr) or OxZr. The proximal head penetration (wear) was measured with repeated RSA examinations over five years. Clinical outcome was measured using the Harris hip score. There was no difference in polyethylene wear between the two head materials when used with either of the two types of acetabular component (p = 0.3 to 0.6). When comparing the two types of polyethylene there was a significant difference in favour of HXLPE, regardless of the head material used (p < 0.001). In conclusion, we found no advantage of OxZr over CoCr in terms of polyethylene wear after five years of follow-up. Our findings do not support laboratory results which have shown a reduced rate of wear with OxZr. They do however add to the evidence on the better resistance to wear of HXLPE over CPE.

Age-related changes in the cholinergic components within the central nervous system. II. Working memory impairment and its relation to hippocampal muscarinic receptors.

Cognitive performance in aging Wistar rats was monitored using the radial arm maze and the latter was correlated with the density of muscarinic receptors in the CNS, using quantitative in vitro receptor autoradiography. Significant working memory deficits were observed in 12, 17 and 24-month-old rats as compared to 3-month-old animals. In addition, the number of the muscarinic receptors declined significantly with age (from 27 to 42% depending on the brain region sampled) utilising [3H]QNB and [3H]PZ receptor binding assays. The above trend became evident already at the age of 12 months. The present findings support the association of central cholinergic activity with memory processes.

Age-related changes in the cholinergic components within the central nervous system of CW1 female mice. I. Structural analysis.

Histomorphometric analysis of age-related structural changes in the brain was performed in CW1 female mice, 3, 9, 24 and 32 months of age. Cholinergic regions, such as the hippocampus, NBM and the medial habenula (MH) were investigated in more detail focusing on morphological parameters. The thickness of the frontoparietal cortex (FPC), and the surface area of the dorsal hippocampus and the MH were found to decrease significantly from 9 to 24 months of age. Except for the unique appearance of pseudo-cysts within the FPC, the structural changes culminated by 24 months. Cells' degeneration, in the CA3 hippocampal subfield, was noted already by 9 months of age whereas in other regions the cells' surface area decreased only between 9 and 24 months. Lipofuscin accumulation was most pronounced in the large neurons of the cortex, hippocampus and NBM at 24 months of age.

Inhibition of haloperidol-induced catalepsy by cholecystokinin octapeptides after central administration to rats.

The possible cataleptogenic or anticataleptic effects of cholecystokinin octapeptide sulphate ester (CCK-8-SE) and desulphated CCK-8 (CCK-8-NS) were evaluated in rats after intracerebroventricular (i.c.v.) administration. Neither CCK-8-SE nor CCK-8-NS induced any sign of catalepsy. When haloperidol, in a dose of 1.0 mg/kg (i.p.), and either CCK-8-SE or CCK-8-NS, in doses of 8, 80 or 800 pmol (i.c.v.), were applied at the same time, the peptides significantly decreased the total duration of catalepsy, the 80 pmol dose being the most effective. For the inhibition of catalepsy CCK-8-NS was the more active as it decreased catalepsy scores even 120 min after administration. Both peptides showed only transient effects on the fully developed catalepsy induced by haloperidol.

Inhibition of seizures induced by picrotoxin and electroshock by cholecystokinin octapeptides and their fragments in rats after intracerebroventricular administration.

The anticonvulsive activity of cholecystokinin octapeptide sulphate ester (CCK-8-SE), non-sulphated cholecystokinin octapeptide (CCK-8-NS) and three different N- and C-terminal fragments were investigated against seizures induced by picrotoxin and electroshock in rats after intracerebroventricular administration. Doses of 0.8 and 80 pmol of CCK-8-SE and CCK-8-NS significantly enhanced the latency of seizures induced by picrotoxin and shortened the duration of the clonic phase of the seizures induced by electroshock. Only CCK-8-SE shortened the recovery time and only 0.8 pmol of CCK-8-SE could shorten the duration of the tonic phase of convulsions induced by electroshock. Doses of the octapeptides of 8000 pmol were ineffective, with the exception of CCK-8-NS in the picrotoxin test. Of the fragments tested, the C-terminal tetrapeptide, CCK-5-8, enhanced the latency of seizures induced by picrotoxin in a dose of 0.8 pmol, and had a dose-dependent biphasic effect on the duration of the clonic phase of seizures induced by electroshock. Intracerebroventricular administration of diazepam enhanced only the latency of tremor and clonic seizures induced with picrotoxin in a dose of 40 nmol. Twelve nmole of diazepam shortened the clonic phase of convulsions induced by electroshock. The peptides tested were much more active than diazepam, and their effective doses were comparable to the amounts of cholecystokinin octapeptide found in brain structures.

Structure-activity and dose-effect relationships of the antagonism of picrotoxin-induced seizures by cholecystokinin, fragments and analogues of cholecystokinin in mice.

Intraperitoneal administration of cholecystokinin octapeptide sulphate ester (CCK-8-SE) and nonsulphated cholecystokinin octapeptide (CCK-8-NS) enhanced the latency of seizures induced by picrotoxin in mice. Experiments with N- and C-terminal fragments revealed that the C-terminal tetrapeptide (CCK-5-8) was the active centre of the CCK octapeptide molecule. The analogues CCK-8-SE and CCK-8-NS (dose range 0.2-6.4 mumol/kg) and caerulein dose range 0.1-0.8 mumol/kg) showed bell-shaped dose-effect curves, with the greatest maximum inhibition for CCK-8-NS. The peptide CCK-5-8 had weak anticonvulsant activity in comparison to the octapeptides, 3.2 mumol/kg and larger doses of the reference drug, diazepam, totally prevented picrotoxin-induced seizures and mortality. The maximum effect of the peptides tested was less than that of diazepam. Experiments with analogues and derivatives of CCK-5-8 demonstrated that the effectiveness of the beta-alanyl derivatives of CCK-5-8 were enhanced and that they were equipotent with CCK-8-SE. Of the CCK-2-8 analogues, Ser(SO3H)7-Ac-CCK-2-8-SE and Thr(SO3H)7-Ac-CCK-2-8-SE and Hyp(SO3H)-Ac-CCK-2-8-SE were slightly more active than CCK-8-SE.

Synthesis of potent heptapeptide analogues of cholecystokinin.

Nine new analogues of acetyl-CCK-heptapeptide (Ac-Tyr(SO3H)2-Met3-Gly4-Trp5-Met6-Asp7-Phe8-NH2 ) were synthesized by solid-phase methodology. In a first series, the Asp7 residue was replaced by hydroxy amino acid sulfate esters. In another series, Gly4 was substituted by D-Ala, while Trp5 and Met6 were replaced by their D enantiomer. The introduction of the sulfate ester was performed with a new, mild, crystalline, and stable reagent, pyridinium acetyl sulfate. Each analogue that contained Tyr(SO3H)2 and a hydroxy amino acid sulfate ester [Ser(SO3H), Thr(SO3H), or Hyp(SO3H)] in position 7 proved to be more potent (1.9, 1.7, and 3.0 times, respectively) than CCK-8 in vitro (isolated gallbladder strips). While devoid of gastrin-like activity in vivo, these analogues had potent anticonvulsive activity. The analogues containing a D-amino acid residue were less potent than the parent compound in vitro. The D-Ala4 replacement, however, yielded a compound that was 40% as potent as CCK-8 in the in vitro test but showed prolonged duration of action on sphincter Oddi. While the 7-substituted Ac-CCK heptapeptides are among the most potent CCK analogues reported so far, the D-Ala4 replacement resulted, for the first time, in prolonged activity in vivo.

Aging, stress, and cognitive function.

Stress was implied as involved in "enhanced aging," and prolonged administration of corticosterone was claimed to lead to central neuronal lesions. This study describes an animal model that simulates the steroid elevation associated with stress by a continuous slow-release administration of corticosterone, in young (3 months old) and middle-aged (12 months old) Fischer 344 rats. Plasma concentrations of corticosterone were stable throughout the day, with no diurnal variation, within the range associated with mild stress. Corticosterone prolonged treatment resulted in morphological changes mainly in the CA1, CA4, and dentate gyrus areas of the hippocampus. Middle-aged rats showed higher vulnerability to the long-term COR treatment than young ones, even when COR treatment was prolonged in young rats from 63 to 90 days. Middle-aged rats were screened before the corticosterone treatment, using the Morris water maze, and divided between cognitively "impaired" and "nonimpaired" subpopulations. Severe cognitive damage during acquisition of the eight-arm radial maze was shown, after the continuous hormonal treatment, in rats initially defined as "nonimpaired" in the Morris water maze. This animal model might be useful for testing the protective effects of drugs against brain changes and cognitive damage, during either pathological or normal aging.

In vivo sulfation of cholecystokinin octapeptide. Possible interactions of the two forms of cholecystokinin with dopamine in the brain.

In most laboratories CCK-8(s) has been found to be the biologically active form of CCK-8 in the CNS. The role of CCK-8(ns) has scarcely been investigated and is poorly understood. CCK-8(s) exerts a transmitter and/or modulator role in this projection. CCK-8(ns), on the other hand, profoundly affects DA-ergic neurotransmission in the nigrostriatal DA-ergic projection. The octapeptide modulates the turnover and release of DA from this neuron population. DA-mediated behavioral reactions are also modulated by CCK-8(ns). We should emphasize that the biological importance of CCK-8(ns) in the CNS has hitherto generally been neglected. Our results point to the equivalence of CCK-8(s) and CCK-8(ns) in the CNS in most biological tests. In some cases the latter compound is the more potent one. In most of these tests the C-terminal fragment (tetragastrin = CCK-4) also proved to be active. It is most likely that a brain receptor population exists which can bind both forms of CCK-8 and even CCK-4. Nevertheless, the CNS could contain binding sites which bind only CCK-8(s) as a ligand. We have found that an unidentified sulfotransferase of the brain can sulfate CCK-8(ns) and thereby provide a ligand for the special receptors of CCK-8(s). It is likely that CCK modulates the turnover and release of DA, and vice versa. Theoretically, different biochemical mechanisms could exist for interactions between CCK octapeptides and DA. We have focused our investigations on the enzymic sulfation-desulfation processes of both CCK-8 and DA and have devised a hypothetical model for the possible interactions. Both CCK-8(ns) and DA could be sulfated in vivo, this enzymic reaction generally requiring active sulfate (PAPS). These two compounds could compete for the limited pool of PAPS, and thus CCK-8 and DA could mutually regulate their levels in the same cell by influencing one of the metabolic (DA) or synthetic (CCK-8(s)) pathways. CCK-8(s) also might provide the O-sulfate group for DA by enzymic transformation, and, conversely, DA-O-sulfate may sulfate CCK-8(ns) in a similar way. These trans-sulfation processes could also mutually determine the concentrations of DA and CCK-8 co-existing in one cell. Experiments to prove these models are planned.

Multiple treatment potentiates the anticonvulsive activity of cholecystokinin octapeptides.

The dose-response curves for the anticonvulsive activity of sulfated and nonsulfated cholecystokinin octapeptide (CCK-8-SE and CCK-8-NS) against picrotoxin-induced (6 mg/kg SC) seizures were assessed either following or without pretreatment with a single high dose of CCK-8-SE or CCK-8-NS, to examine acute tolerance to the effect after IP injections in mice. As CCK-8-SE or CCK-8-NS pretreatment, a 1.6 mumole/kg dose was injected 2 hr prior to the second injection. No acute tolerance to the anticonvulsive activity was demonstrated, and CCK-8-NS pretreatment significantly potentiated its own anticonvulsive activity. Chronic (8-day) daily treatment with a 0.16 mumole/kg dose of CCK-8-SE or CCK-8-NS antagonized seizures by picrotoxin, presumably in a cumulative manner. To investigate the interactions of CCK octapeptides with other anticonvulsive agents, picrotoxin-induced seizures were antagonized with several doses of diazepam following or without acute, high-dose pretreatment with CCK-8-SE or CCK-8-NS. The two octapeptides only slightly modified the activity of diazepam: CCK-8-SE pretreatment displayed a tendency to antagonize it, while CCK-8-NS pretreatment to potentiate it. The results suggest that multiple treatment with CCK-8 induces sensitization of CCK receptors mediating anticonvulsive activity.

Neuropharmacological actions of the superactive agonist analog D-TRP-6-LH-RH after peripheral injection into mice.

The neuropharmacological actions of the agonist analog D-Trp-6-LH-RH were investigated in several tests after subcutaneous administrations to male mice. The doses applied were in the range 1-1000 micrograms/kg. D-Trp-6-LH-RH doses of 10 micrograms/kg and higher induced significant analgesic effects in the hot-plate and tail-flick tests, and decreased the open-field parameters (ambulation, rearing, grooming). The 100 and 1000 micrograms/kg doses increased the latencies of picrotoxin-induced seizures, significantly inhibited apomorphine-induced cage climbing and also exerted a cataleptogenic effect. The results indicate that this agonist analog of LH-RH has an inhibitory effect on the central nervous system, and the mechanism of its action may involve dopaminergic transmission and/or endogenous opiates.

The effects of sulfated and nonsulfated cholecystokinin octapeptides on electroconvulsive shock-induced retrograde amnesia after intracerebroventricular administration in rats.

The effects of several doses of intracerebroventricularly injected cholecystokinin octapeptide sulfate ester (CCK-8-SE) and nonsulfated scholecystokinin octapeptide (CCK-8-NS) were studied on electroconvulsive shock (ECS)-induced retrograde amnesia, as measured in a one-trial step-through passive avoidance paradigm. Both CCK-8-SE and CCK-8-NS were able to attenuate amnesia slightly when they were injected into rats 10 min prior to ECS treatment, possibly by reducing the severity of the ECS-induced seizures. Of the treatments carried out immediately after ECS, only the 0.8 pmole dose of CCK-8-NS could significantly restore retrograde amnesia. After treatment 20 min prior to testing 24-hr retention, no effect of the peptides was observed. The lack of a dose-dependency and of any effect on retrieval raises the possibility that the CCK octapeptides influence memory processes by an indirect mechanism.

Inhibition of feeding by the C-terminal tetrapeptide fragment of cholecystokinin in a novel environment.

Several doses of the C-terminal tetrapeptide fragment of cholecystokinin octapeptide (CCK-5-8) were injected intraperitoneally (ip.) and intracerebroventricularly (icv.), and their effects on the feeding of 24-hr food-deprived rats in familiar and novel environments were studied. In the familiar environment, CCK-5-8 ip. or icv. had no effect on the food intake of the rats. In the novel environment (i.e. in an open field), CCK-5-8 showed merely slight effects on the main open-field parameters: only ip. administered CCK-5-8 enhanced the incidence of grooming. Of the feeding parameters tested in the open field, mainly the food intake/approach to food ratio was depressed by CCK-5-8 ip. or icv. The 24 mumole/kg ip. dose of CCK-5-8 decreased the food intake, while the 8 pmole icv. dose increased the number of approaches to food. After the treatments found to be the most effective, the latency to first bite was also enhanced. The results suggest that CCK-5-8 can amplify the arousal enhancement elicited by novelty through a central mechanism.

Cataleptogenic and anticataleptic activity produced by cholecystokinin octapeptides in mice.

The catalepsy induced by subcutaneously (sc.) and intracerebroventricularly (icv.) administered cholecystokinin octapeptide sulfate ester (CCK-8-SE) and desulfated cholecystokinin octapeptide (CCK-8-NS), and the effects of CCK-8-SE and CCK-8-NS on haloperidol-induced catalepsy, were investigated in mice. The results demonstrate the bimodal effect of CCK octapeptides in a catalepsy test. With sc. administration CCK-8-SE in the doses of 0.4 or 0.8 mumole/kg, but not CCK-8-NS at any dose, induced catalepsy. Furthermore, the catalepsy induced by CCK-8-SE was of short duration. With icv. administration only 40 pmole CCK-8-NS induced significant catalepsy. When 0.2, 0.4 and 0.8 mumole/kg sc. doses of CCK-8-NS or 0.4 pmole icv. dose of CCK-8-SE or CCK-8-NS was given in combination with intraperitoneal (ip.) administration of 1.0 mg/kg haloperidol, the total duration of catalepsy was suppressed. Finally, CCK-8-SE sc. when given in combination with haloperidol ip., exerted a biphasic, synergistic-antagonistic effect on the haloperidol-induced catalepsy.

Nimodipine's protection against corticosterone-induced morphological changes in the hippocampus of young rats.

Sustained high levels of corticosterone (CORT), one of the major stress-induced hormones in the rat, were suggested as generating 'accelerated brain aging' and were shown to induce both specific brain changes in the hippocampus and learning impairments in young and middle-aged Fischer-344 rats. Evidence that altered calcium (Ca) homeostasis may play a major role in brain aging has accumulated over the last decade. Recently, new data established a connection between glucocorticoids and voltage-activated Ca influx in aged hippocampal neurons. In the present study, an attempt was made to block the CORT-induced 'accelerated aging' by the simultaneous administration of the L-type Ca channel blocker nimodipine. CORT or placebo sustained-release (SR) pellets were implanted subcutaneously in 3 months old Fischer male rats. Each group was further sub-divided between nimodipine and placebo SR treatments. Characteristic CORT-induced morphological changes were observed in pyramidal hippocampal cells, such as at the CA1 and CA4 sub-regions (22.2% +/- 7.7 and 28.6% +/- 8.4 of pyknotic cells without clear nuclei, respectively). Concomitant treatment with nimodipine conferred full protection against CORT-induced morphological changes (e.g. 3.2% +/- 0.8 and 2.1% +/- 1.9 of pyknotic cells in CA1 and CA4, n = 7 rats in each group; P < 0.04). The neuroprotective efficacy of nimodipine supports the theory of Ca involvement in CORT related 'accelerated brain aging'.

Age-related structural changes in the rat hippocampus: correlation with working memory deficiency.

Age-related histopathological changes in the hippocampal formation were correlated with cognitive performance, evaluated in rats at the 8-arm radial maze. Experiments were conducted using young (3 months), mature (12 months), middle-aged (17 months) and aged (24 months) Wistar rats. Significant memory impairments were already observed at the age of 12 months in all the measured parameters (correct choices, percent errors and total time). No further decline was observed between 12 and 17 months of age, while at 24 months additional decline was monitored mainly in the percent errors parameter. Morphometric analysis revealed a decrease in the area of cells within the hippocampus and the number of cells in the CA3 subfield. This pattern of morphological changes with age corresponded well with the cognitive impairments, with high correlation especially to lesions at the CA3 subfield. It had also been confirmed in this study that lipofuscin appeared to be a good histochemical marker for CNS cell degeneration. It is concluded that 12-month-old Wistar rats may serve as the animal model of choice for the study of specific age-related behavioral deficits and that the hippocampal CA3 region might play a major role in the age-dependent cognitive decline.

The effects of long-term corticosterone administration on hippocampal morphology and cognitive performance of middle-aged rats.

The main objective of this research was to study the relationship between glucocorticoids, aging and the deterioration of cognitive functions. Towards this end, an attempt was made to develop an animal model which will enable the investigation of such interactions, using subcutaneously implanted sustained-release corticosterone pellets. The goal was to achieve moderately high concentrations of corticosterone in plasma, comparable to the peak basal levels or to those found under mild stress. Middle-aged (12 months old) Fischer-344 rats, used in this study, were divided before the prolonged hormonal treatment into cognitively 'impaired' and 'non-impaired' groups using the Morris water maze. The cognitive impairment, which was induced by the long-term corticosterone administration, was exhibited during acquisition of the 8-arm radial maze only in the 'non-impaired' group. Behavioral scores for drug-treated 'impaired' rats were not statistically different from those of the placebo-treated 'impaired' group. The morphological deterioration in hippocampal areas of the brain was quantified and revealed high correlation with the behavioral data. This animal model may become extremely useful in testing projected prophylactic therapy against the brain damage and cognitive deficits induced by the high corticosteroid-aging combination.

Dose-dependent effect of nitric oxide synthase inhibition following transient forebrain ischemia in gerbils.

The extensive research concerning the interaction between nitric oxide (NO) and ischemic brain tissue has yielded contradictory results. The present study was designed to explore the effect of gradual inhibition of NO production on brain ischemia. Gerbils were administered (i.p.) either saline (control-ischemia), or 5, 10, 25 or 50 mg/kg of NG-nitro-L-arginine (NARG), a specific inhibitor of NO synthase (NOS), and 4 h later were subjected to 5 min of forebrain ischemia. A group receiving 50 mg/kg NARG with sham operation served as a second control (control-NARG) group. Body weights and spontaneous activity were monitored daily until day 6, when the gerbils were sacrificed and their brains processed for histologic-morphometric evaluation. All ischemia groups displayed significant decreases in body weights starting on day 1, as compared to control-NARG (non-ischemic) gerbils. At 24 h post-ischemia spontaneous activity was increased in all ischemia groups in a dose-dependent manner, reaching a peak at 25 mg/kg. Typical ischemia-induced neuronal cell degeneration was observed at the hippocampal CA1 layer in control-ischemia and in each of the dose-groups of 10 mg/kg NARG and above. The 5 mg/kg group displayed damage which was not different from control-NARG, and was milder (P < 0.01) than control-ischemia gerbils and each of the other dose-groups. It is suggested that during ischemia, NO activates a series of processes which are beneficial to brain tissue, whereas an excess amount of NO causes neurotoxic effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective hippocampal lesion following omega-conotoxin administration in rats.

Histopathological evaluation of rat brains 3 days following unilateral i.c.v. injections of omega-conotoxin GVIA (omega-ctx), 0.032 and 0.1 nmol/kg, was performed. An isolated unilateral lesion confined to the injected hemisphere was found in the hippocampal CA3 neurons. Morphometric analysis of these cells revealed a significant reduction in cell area in both dose groups compared to i.c.v. injected vehicle, and to the contralateral hemisphere. These data indicate a specific degenerative process and suggest that CA3 cells possess omega-ctx-sensitive Ca2+ channels which are essential to their viability.