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A 32-year-old woman, gravida 1, para 0, was referred to our pregnancy ultrasound unit at 20 weeks’ gestation for obstetrics ultrasound because of her cardiac disease history. She was affected by a ...

The effects of metrifonate were investigated in 4-6- and 22-24-month-old rats. Extracellular acetylcholine levels were measured by transversal microdialysis in vivo. Baseline extracellular acetylcholine levels in the cerebral cortex and hippocampus were 42% and 60% lower, respectively, in old than in young rats. Old rats did not discriminate between familiar and novel objects. In old rats, metrifonate (80 mg/kg p.o.) brought about 85% inhibition of cholinesterase activity in the cortex and hippocampus, a 4-fold increase in extracellular acetylcholine levels in the cortex only, and restored object recognition. In young rats, metrifonate caused 75% cholinesterase inhibition in the cerebral cortex and hippocampus, a 2-fold increase in cortical and hippocampal extracellular acetylcholine levels, and no effect on object recognition. The slight cholinesterase inhibition following metrifonate (30 mg/kg) in aged rats had no effect on cortical acetylcholine levels and object recognition. In conclusion, metrifonate may improve the age-associated cholinergic hypofunction and cognitive impairment.

1. The effects of histamine and agents at histamine receptors on spontaneous and 100 mM K(+)-evoked release of acetylcholine, measured by microdialysis from the cortex of freely moving, rats, and on cognitive tests are described. 2. Local administration of histamine (0.1-100 microM) failed to affect spontaneous but inhibited 100 mM K(+)-stimulated release of acetylcholine up to about 50%. The H3 receptor agonists (R)-alpha-methylhistamine (RAMH) (0.1-10 microM), imetit (0.01-10 microM) and immepip (0.01-10 microM) mimicked the effect of histamine. 3. Neither 2-thiazolylethylamine (TEA), an agonist showing some selectivity for H1 receptors, nor the H2 receptor agonist, dimaprit, modified 100 mM K(+)-evoked release of acetylcholine. 4. The inhibitory effect of 100 microM histamine was completely prevented by the highly selective histamine H3 receptor antagonist, clobenpropit but was resistant to antagonism by triprolidine and cimetidine, antagonists at histamine H1 and H2 but not H3 receptors. 5. The H3 receptor-induced inhibition of K(+)-evoked release of acetylcholine was fully sensitive to tetrodotoxin (TTX). 6. The effects of intraperitoneal (i.p.) injection of imetit (5 mg kg-1) and RAMH (5 mg kg-1) were tested on acetylcholine release and short term memory paradigms. Both drugs reduced 100 mM K(+)-evoked release of cortical acetylcholine, and impaired object recognition and a passive avoidance response. 7. These observations provide the first evidence of a regulatory role of histamine H3 receptors on cortical acetylcholine release in vivo. Moreover, they suggest a role for histamine in learning and memory and may have implications for the treatment of degenerative disorders associated with impaired cholinergic function.

Object recognition was investigated in adult and aging male rats in a two-trials, unrewarded, test that assessed a form of working-episodic memory. Exploration time in the first trial, in which two copies of the same object were presented, was recorded. In the second trial, in which one of the familiar objects and a new object were presented, the time spent exploring the two objects was separately recorded and a discrimination index was calculated. Adult rats explored the new object longer than the familiar object when the intertrial time ranged from 1 to 60 min. Rats older than 20 months of age did not discriminate between familiar and new objects. Object discrimination was lost in adult rats after scopolamine (0.2 mg/kg SC) administration and with lesions of the nucleus basalis, resulting in a 40% decrease in cortical ChAT activity. Both aniracetam (25, 50, 100 mg/kg os) and oxiracetam (50 mg/kg os) restored object recognition in aging rats, in rats treated with scopolamine, and with lesions of the nucleus basalis. In the rat, object discrimination appears to depend on the integrity of the cholinergic system, and nootropic drugs can correct its disruption.

The nucleus basalis of male Charles River Wistar rats was injected with 10 micrograms of the beta-amyloid peptides beta-(1-40) and beta-(25-35) and changes in the morphology of the lesioned area, the release of acetylcholine from the cortex, and in behavior were investigated. Injections of saline and a scrambled (25-35) peptide were used as controls. One week after lesioning, a Congo Red-positive deposit of aggregated material was found at the beta-peptides injection site, which lasted for about 21 days in the case of the beta-(25-35) peptide and at least two months for beta-(1-40). No deposit was detected after scrambled peptide injection. At one week post injection, an extensive glial reaction surrounded the injection site of all peptides and saline as well. Such a reaction was still present but rather attenuated after two months. A decrease in the number of cholinergic neurons was detected in the nucleus basalis after one week with all treatments except saline. After two months, a reduction in the number of choline acetyltransferase-immunopositive neurons was still detectable in the rats injected with beta-(1-40) but not in the beta-(25-35)-or scrambled-injected. The reduction in choline acetyltransferase immunoreactivity was closely paralleled by a decrease in basal acetylcholine release from the parietal cortex ipsilateral to the lesion. Disruption of object recognition was observed in the first weeks after beta-(25-35) peptide injection, whereas the beta-(1-40) peptide impaired the performance only two months after lesion. Rats with lesions induced by beta-peptides may be a useful animal model of amyloid deposition for investigation of the pathogenetic mechanisms leading to Alzheimer's disease.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetically transmitted cerebrovascular disease. Typically, the first clinical manifestation is migraine and the full clinical spectrum of the disease with recurrent strokes of the subcortical type, cognitive, and mood disorders is seen during the fourth and fifth decades of life. Vascular risk factors are usually absent in CADASIL patients and the diagnosis of the disease is particularly suspected in young adults with cerebrovascular events of unknown cause, diffuse leukoencephalopathy on computed tomography or magnetic resonance imaging, and a history of cerebrovascular diseases or dementia in many family members. We describe three Italian CADASIL patients who presented to medical attention for cerebrovascular events occurred after the age of 55 and had, in addition to hypertension and hyperlipidemia, thrombophilic risk factors such as hyperhomocysteinemia, elevated levels of lipoprotein(a), and antiphospholipid antibodies. Symptoms possibly related to cortical involvement, such as dysphasia and visual field deficits, were reported by two of these patients. We conclude that a diagnosis of CADASIL should not be disregarded in patients with vascular risk factors and presenting with symptoms not immediately referable to subcortical damage at ages more advanced than commonly reported.

The relation between chronic cerebral hypoperfusion and cognitive functions has not been completely clarified. The resolution of cerebral hypoperfusion states, such as those induced by arteriovenous malformations or carotid stenosis/occlusion, has been reported to improve mental decline in humans. Subcortical vascular dementia is another human condition supposed to be linked with chronic cerebral hypoxia/ischemia. The extent of this cause/effect relation is, however, difficult to be assessed in humans, where different factors, such as ageing or subtle degenerative processes, can coexist and interact influencing cognitive performances. Experimental studies can help to elucidate this relation because they can use models of pure chronic/moderate cerebral hypoperfusion. An experimental model of chronic ischemia is the bilateral common carotid artery occlusion in the rat. In this paper, we present a review of experimental studies that evaluated cognitive functions in the rat with bilateral common carotid artery occlusion. We then present an experimental model of bilateral common carotid artery occlusion in the rat modified with respect to previous papers regarding both the surgical procedure and the neurocognitive evaluation that is focused on cognitive domains depending on subcortical-frontal circuits. We propose this model to investigate subcortical vascular dementia.

The effects of 21-day treatment with the acetylcholinesterase inhibitors metrifonate (80 mg kg−1 per os (p.o.)) and tacrine (3 mg kg−1 p.o.), twice daily, on cortical and hippocampal cholinergic systems were investigated in aged rats (24–26 months). Extracellular acetylcholine levels were measured by transversal microdialysis in vivo; choline acetyltransferase and acetylcholinesterase activities were measured ex vivo by means of radiometric methods. Basal cortical and hippocampal extracellular acetylcholine levels, measured 18 h after the last metrifonate treatment, were about 15 and two folds higher, respectively, than in control and tacrine-treated rats. A challenge with metrifonate further increased cortical and hippocampal acetylcholine levels by about three and four times, respectively. Basal extracellular acetylcholine levels, measured 18 h after the last treatment with tacrine were not statistically different from those of the control rats. A challenge with tacrine increased cortical and hippocampal extracellular acetylcholine levels by about four and two times. A 75% inhibition of cholinesterase activity was found 18 h after the last metrifonate administration, while only a 15% inhibition was detectable 18 h after the last tacrine administration. The challenge with metrifonate or tacrine resulted in 90 and 80% cholinesterase inhibition, respectively. These results demonstrate that in aging rats a subchronic treatment with metrifonate results in a long-lasting, cholinesterase inhibition, and a persistent increase in acetylcholine extracellular levels which compensate for the age-associated cholinergic hypofunction. Metrifonate is therefore a potentially useful agent for the cholinergic deficit accompanying Alzheimer's disease.

Publisher Summary This chapter focuses on the actions of β-peptides in the nucleus basalis (NB) at longer times post-injection. The effects of β-peptides on acetyltransferase (ChAT) immunoreactivity (IR), cortical acetylcholine (ACh) release and behavior are examined. The chapter investigates the toxicity of β-peptides to the cholinergic neurons by direct injection in the forebrain of the rat. As it is reported that the neurotoxic activity of β-amyloid resides within the 25–35 portion of the peptide, the full-length peptide β and the β-peptide are studied. The chapter demonstrates that the administration of β-peptides into the medial septum is followed by a decrease in ACh release from the hippocampus. The chapter also studies the histological and functional changes due to the presence in the NB of β-peptide deposits. It concludes that local injections of amyloid peptides in the rat NB result in local damage and cholinergic hypofunction. However, only the β-(1-40) and β-(25-35) peptides, but not the scrambled form congophilic fibrillary deposits, and induce some behavioral impairment, with the most persistent decrease in the number of cholinergic neurons and ACh release brought about by β-(1- 40). Because the deposit formed by the β-(l-40) peptide lasts largely unaltered for a long time, the injection of this peptide may be a useful model for investigating the temporal progression and neurotoxic effects of an amyloid plaque.

Male Wistar rats (3- and 20-month-old) were perfused i.c.v. with 1.5 micrograms of either nerve growth factor (NGF) or cytochrome C daily for 14 days. At the end of the infusion, the object-recognition test was carried out and extracellular acetylcholine levels (ACh) were measured in the cortex and hippocampus by transversal microdialysis technique. In 20-month-old control rats, the cortical and hippocampal ACh levels were 35 and 45% lower, respectively, than in 3-month-old rats and the ability to discriminate between a familiar and new object was impared. In the old rats treated with NGF, the ACh release as well as the behavioral performance showed no difference from those of young rats. These findings indicate that both ACh levels and memory impairment are improved in aged rats by NGF treatment and suggest that there is a relationship between object recognition and the activity of the forebrain cholinergic system.

The extent and duration of cholinergic hypofunction induced by long-term ethanol consumption was investigated in the rat. Ethanol (20% v/v) was administered to male adult Wistar rats as the sole source of fluid for three or six months. Control rats received tap water. The body weight, food and fluid intake in ethanol-treated rats were lower than in control rats throughout the treatment. After three months of ethanol consumption, and one week withdrawal, acetylcholine release in freely moving rats, investigated by microdialysis technique coupled to high-performance liquid chromotagraphy quantification, was significantly decreased by 57 and 32% in the hippocampus and cortex, respectively, while choline acetyltransferase activity was significantly decreased (−30%) only in the hippocampus. A complete recovery of choline acetyltransferase activity and acetylcholine release was found after four ethanol-free weeks. Conversely, after four weeks of withdrawal following six months of ethanol treatment, the recovery in acetylcholine release was not accompanied by that in choline acetyltransferase activity, which remained significantly lower than in control rats in both cortex and hippocampus. The ability of rats to negotiate active and passive avoidance conditioned response tasks, tested after four ethanol-free weeks, was strongly impaired in both three- and six-month ethanol-treated rats. In conclusion, our experiments demonstrate that the development of a long-lasting cholingergic hypofunction requires at least six months of ethanol administration. The hypofunction affects choline acetyltransferase activity and acetylcholine release differently, and undergoes a remarkable recovery.

To the Editor: Strong and reliable outcome measures are required in laboratory studies that aim to appraise the extent of the damage in animals subjected to various forms of cerebral ischemia. In this regard, the assessment of histological changes, such as the volume of infarcted tissue or the number of necrotic cells, is considered the gold standard. However, the assessment of functional outcome can also be useful in animal studies that evaluate the effect of new therapeutic agents, since the clinical examination is effortless and not time demanding; moreover, physical testing of the animals can be repeated over time and thus provide data on the evolution of the neurological deficit. A simple neurological score to evaluate sensorimotor performance in rats has recently been developed by Garcia et al.1 It explores six different functions and attributes to each a 3- or 4-point score. The total score, which correlates closely with the severity of the histological injury (in particular with the number of necrotic neurons) in a model of middle cerebral artery (MCA) occlusion in the Wistar rat,1 has been used as one of the outcome measures in studies that estimate the effect of new drugs.2 3 Because of its simplicity and strong correlation with histological damage, this score might be used in other laboratories employing experimental procedures to cause cerebral ischemia. In this regard, evaluation of its reproducibility could be extremely useful, because it is known that the validity of scales evaluating neurological deficit can be affected by interobserver variability.4 5 We have conducted a study to evaluate the impact of personal judgment in the …

The effects of GM1 ganglioside (30 mg/kg i.p.) administration for 22 days on choline acetyltransferase (ChAT) activity and noradrenaline (NA) levels in the cerebral cortex and on the acquisition of active and passive avoidance-conditioned responses were investigated in both sham-operated rats and in rats with a unilateral electrolytic lesion of the magnocellular forebrain nuclei (MFN). A statistically significant ChAT decrease in cortical areas ipsilateral to the lesion was found in saline-treated lesioned rats. In the lesioned GM1-treated rats, ChAT activity was only reduced in the frontoparietal areas and was significantly increased in the ipsilateral parieto-occipital areas as well as in both contralateral regions. NA levels in the cortex were neither significantly affected by the lesion nor by GM1 treatment. The lesion impaired the acquisition of active and passive conditioned avoidance responses. GM1 treatment improved acquisition of the active avoidance response in the lesioned rats as indicated by a larger number of avoidances and a smaller number of escape failures during training in comparison with saline treatment. Ganglioside had no effect on the passive avoidance responses. These results demonstrate that GM1 administration facilitates the recovery of the cortical cholinergic system and of behavioral responses impaired by an electrolytic lesion of the cholinergic forebrain nuclei.

The neurochemical and behavioural recovery following unilateral and bilateral lesions of the nucleus basalis was investigated in adult male Wistar rats 20 days and 6 months after surgery. The lesions were made by stereotaxic injections of ibotenic acid. Twenty days after surgery there was a statistically significant choline acetyltransferase decrease in the frontal and parietal ipsilateral cortex of the unilaterally lesioned rats and in the cortex of both hemispheres after bilateral lesions. Cortical high affinity choline uptake rate was significantly decreased 4 days after lesions but showed a rapid recovery within 20 days post-lesion in unilaterally and bilaterally lesioned rats. However, at this time both groups of lesioned rats showed a marked impairment in the acquisition of passive and active (shuttle-box) avoidance conditioned responses. Six months after surgery the decrease in choline acetyltransferase activity was smaller and statistically significant in the ipsilateral frontal cortex only in the unilaterally lesioned rats and in the frontal and parietal cortex of both hemispheres in the bilaterally lesioned rats. High affinity choline uptake was increased in the contralateral hemispheres of the unilaterally lesioned rats and was significantly larger than in the bilaterally lesioned rats. There was no difference in the acquisition of both passive and active avoidance conditioned responses between the sham operated and unilaterally lesioned rats, while the bilaterally lesioned rats could only negotiate the active avoidance conditioned response. In conclusion, our experiments demonstrate a remarkable neurochemical and behavioural recovery within 6 months in rats with a unilateral lesion of the nucleus basalis and only a limited recovery in the bilateral lesioned rats.

The intracerebroventricular injection of 0.5 mg of vincristine sulphate in adult male Wistar rats caused within 11 days the impairment of motor and reflexive behavior, evaluated by the elevated platform and hanging wire tests, a decrease in food consumption and loss of body weight, a 45% decrease in hippocampal choline acetyltransferase (ChAT) activity and a 35% decrease in the rate of high-affinity choline uptake (HACU) in the injected side. The latter effects are due to the death of neurons in the respective hemiseptum. Intrafimbrial injection of vincristine caused the same decrease in ChAT activity without behavioral alterations. Daily i.p. administration of GM1 ganglioside, beginning immediately after the vincristine injection, prevented dose dependently the decrease in ChAT activity and HACU rate. Prevention was complete with the 60 mg/kg dose. The same dose was equally active on ChAT activity when given s.c. but was inactive p.o. The ChAT decrease was also prevented when GM1 treatment began 5 days after vincristine. GM1 60 mg/kg i.p. also reduced the behavioral toxicity of vincristine. The possibility that GM1 might prevent vincristine toxicity by antagonizing its disruption of neurofilaments and axonal flow is discussed.

Lecithin:cholesterol acyltransferase (LCAT) activity has been examined in the rat by using a brain homogenate preparation as the phospholipid substrate and blood plasma as the enzyme source. LCAT activity was detected on using 60 mul of serum onwards. Successive experiments have also shown that LCAT activity is present in the edematous rat brain tissue homogenate when incubated with inactivated rat plasma as substrate. The results are discussed in relation to cholesteryl ester accumulation in brain during demyelinating diseases.

Several studies have been carried out in recent years on the chemical modification of brain white matter lipids in various pathological conditions, and particular attention has been given to “demyelination” occurring in inflammatory, traumatic, vascular and tumoral conditions. Many evidences indicate, as a most significative result, the increase of cholesterol esters in nervous tissue. These compounds, normally absent in adult brain tissue, increase in fact drammatically either in demyelinated areas or in apparently normal white matter, as already indicated long ago by Cumings (1953;1955). For this reason cholesterol ester metabolism has been largely studied, either in normal growing brain (Pritchard, 1964) and in normal adult brain or in pathological brain tissue.

Summary Background Trabectedin is an alkylating drug with a unique mechanism of action causing single-strand and double-strand DNA breaks that activate DNA damage-response pathways. Based on our preclinical data, we hypothesised that poly(ADP-ribose) polymerase 1 (PARP1) inhibitors might be an ideal partner of trabectedin and aimed to assess the safety, identify the recommended phase 2 dose, and explore preliminary signs of activity of trabectedin and olaparib combination treatment in patients with bone and soft-tissue sarcoma. Methods We did an open-label, multicentre, phase 1b study, recruiting patients from the national Italian sarcoma network aged 18 years and older with histologically confirmed bone and soft-tissue sarcoma progressing after standard treatments with Eastern Cooperative Oncology Group performance status of 1 or less. In a classic 3 + 3 design, patients received a 24 h infusion of trabectedin on day 1 and olaparib orally twice a day in 21-day cycles across six dose levels (trabectedin 0·675–1·3 mg/m2 every 3 weeks; olaparib 100–300 mg twice a day from day 1 to 21). Intermediate dose levels were permitted to improve safety and tolerability. The primary endpoint was determination of the recommended phase 2 dose (the maximum tolerated dose). Safety and antitumour activity were assessed in all patients who received at least one dose of the study drugs. We report the results of the dose-escalation and dose-expansion cohorts. The trial is still active but closed to enrolment, and follow-up for patients who completed treatment is ongoing. This trial is registered with ClinicalTrials.gov, number NCT02398058. Findings Between Nov 17, 2014, and Jan 30, 2017, of 54 patients assessed for eligibility, we enrolled 50 patients: 28 patients in the dose-escalation cohort and 22 patients in the dose-expansion cohort. Patients received a median of four cycles of treatment (IQR 2–6; range 1–17 [the patients who received the highest number of cycles are still on treatment]) with a median follow-up of 10 months (IQR 5–23). Considering all dose levels, the most common grade 3–4 adverse events were lymphopenia (32 [64%] of 50 patients), neutropenia (31 [62%]), thrombocytopenia (14 [28%]), anaemia (13 [26%]), hypophosphataemia (20 [40%]), and alanine aminotransferase concentration increase (9 [18%]). No treatment-related life-threatening adverse events or deaths occurred. One (2%) patient interrupted treatment without progression without reporting any specific toxicity. Observed dose-limiting toxicities were thrombocytopenia, neutropenia for more than 7 days, and febrile neutropenia. We selected intermediate dose level 4b (trabectedin 1·1 mg/m2 every 3 weeks plus olaparib 150 mg twice a day) as the recommended phase 2 dose. Seven (14%; 95% CI 6–27) of 50 patients achieved a partial response according to Response Evaluation Criteria In Solid Tumors 1.1. Interpretation Trabectedin and olaparib in combination showed manageable toxicities at active dose levels for both drugs. Preliminary data on antitumour activity are encouraging. Two dedicated phase 2 studies are planned to assess activity of this combination in both ovarian cancer (EudraCT2018-000230-35) and soft-tissue sarcomas. Funding Italian Association for Cancer Research, Italian Sarcoma Group, Foundation for Research on Musculoskeletal and Rare Tumors, and Italian Ministry of Health.