The specific PKR inhibitor C16 prevents apoptosis and IL-1ß production in an acute excitotoxic rat model with a neuroinflammatory component.

The double-stranded RNA-dependent protein kinase (PKR), an apoptotic inducer, regulates much pro-inflammatory cytokine production. The purpose of this study was to evaluate in vivo the effects of the specific PKR inhibitor C16 in the striatum in an acute excitotoxic rat model with an important neuroinflammatory component. Inflammation was induced by unilateral striatal injection of quinolinic acid (QA) in 10-week-old normotensive rats. Animals were separated into groups receiving either vehicle or C16 for both sham and QA rats. The effects were assessed in ipsi- and contralateral striata by immunoblotting for PKR activation, by Luminex assay for cytokine levels and by immunofluorescent staining for cleaved caspase-3 to detect neuronal apoptosis. The highest dose of C16 (600µg/kg; C16-2) in QA rats reduced expression of the active catalytic domain of the PKR vs. that in vehicle-injected QA rats. A robust increase of IL-1ß levels on the contralateral side of QA rats was prevented by C16-2 (97% inhibition). Macroscopic and microscopic observation of cerebral tissue (Hematoxylin & Eosin staining) revealed that tissue integrity was more preserved with C16-2 treatment than its vehicle in QA rats. Furthermore, C16-2 treatment decreased by 47% the neuronal loss and by 37% the number of positive cleaved caspase-3 neurons induced by QA injection. In conclusion, C16 prevented not only the PKR-induced neuronal loss but also the inflammatory response in this acute excitotoxic in vivo model, highlighting its promising neuroprotective properties to rescue acute brain lesions.

Basal ganglia neuroprotection with anticonvulsants after energy stress: a comparative study.

The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model provides a valuable paradigm of the energy deficiency disorders found in childhood. In such disorders, anticonvulsants may provide neuroprotection by modulating cellular energy consumption and by exerting favorable pleiotropic effects on neuronal survival. To verify such hypothesis, we tested the effects of levetiracetam, vigabatrin, gabapentine, pregabaline, tiagabine, clonazepam and lamotrigine on neuroprotection in the MPTP mouse model. The membrane dopamine transporter (DAT) density, which provides a reliable index of dopaminergic neurons survival in the basal ganglia, was assessed by semi-quantitative autoradiography of the striatum. Unlike all other anticonvulsants tested, lamotrigine provided a significant and dose-dependent neuroprotection in these experimental conditions. Lamotrigine, a widely used and well-tolerated molecule in children, could provide neuroprotection in various energy deficiency disorders.

MPTP intoxication in mice: a useful model of Leigh syndrome to study mitochondrial diseases in childhood.

The basal ganglia, which are interconnected in the striato-nigral dopaminergic network, are affected in several childhood diseases including Leigh syndrome (LS). LS is the most common mitochondrial disorder affecting children and usually arise from inhibition of the respiratory chain. This vulnerability is attributed to a particular susceptibility to energetic stress, with mitochondrial inhibition as a common pathogenic pathway. In this study we developed a LS model for neuroprotection trials in mice by using the complex I inhibitor MPTP. We first verified that MPTP significantly inhibits the mitochondrial complex I in the brain (p = 0.018). This model also reproduced the biochemical and pathological features of LS: MPTP increased plasmatic lactate levels (p = 0.023) and triggered basal ganglia degeneration, as evaluated through dopamine transporter (DAT) autoradiography, tyrosine hydroxylase (TH) immunohistochemistry, and dopamine dosage. Striatal DAT levels were markedly decreased after MPTP treatment (p = 0.003). TH immunoreactivity was reduced in the striatum and substantia nigra (p = 0.005), and striatal dopamine was significantly reduced (p < 0.01). Taken together, these results confirm that acute MPTP intoxication in young mice provides a reproducible pharmacological paradigm of LS, thus opening new avenues for neuroprotection research.

n-3 polyunsaturated fatty acid supplementation reverses stress-induced modifications on brain monoamine levels in mice.

The aim of this study was to examine the effects of supplementation with n-3 polyunsaturated fatty acids (PUFAs) on stress responses in mice subjected to an unpredictable chronic mild stress (UCMS) procedure. Stress-induced modifications in coat and aggressiveness were evaluated, and phospholipid PUFA profiles and monoamine levels were analyzed in the frontal cortex, hippocampus, and striatum. The results showed that repeated exposure to mild stressors induced degradation in the physical state of the coat, lowered body weight gain, and increased aggressiveness, without any effect of n-3 PUFA supplementation. The UCMS induced a significant decrease in the levels of norepinephrine in the frontal cortex and striatum, and a nonsignificant decrease in the hippocampus. The tissue levels of serotonin (5-HT) were 40% to 65% decreased in the three brain regions studied. Interestingly, the n-3 PUFA supplementation reversed this stress-induced reduction in 5-HT levels. These findings showed that supplementation in n-3 long-chain PUFAs might reverse certain effects of UCMS in cerebral structures involved in stress-related behaviors.

[Postradiation dental extractions]. / Extractions dentaires en territoire irradié

INTRODUCTION: Healing may fail after postradiation dental extractions, with a risk of followed osteoradionecrosis, and its dramatic prognosis. We propose a protocol for postradiation extractions. MATERIAL AND METHODS: From January 2000 to December 2001, 107 patients underwent 287 dental extractions after radiation therapy: 80.5% in mandibular jaw, average radiation dose 61.6 Gy, average time between radiation and extraction 6 years. Extractions were performed under general anesthesia, diazanalgesia or local anesthesia, and extraction wounds were sutured after insertion of a collagenic sealant impregnated with gentamicin. General antibiotic prophylaxy was given. RESULTS: Healing failed in only two cases. The first case involved early-stage osteoradionecrosis and the second a tumoral localization. The rate of post-extraction osteoradionecrosis was thus 0.35%. DISCUSSION: A strict protocol associating non-traumatic surgery and an alveolar sealant can considerably decrease the risk of osteoradionecrosis after dental extraction in radiated bone.

mRNA D(2) dopaminergic receptor expression after hypoxia-ischemia in rat immature brain.

Previous studies have shown a reduction of dopaminergic D(2) receptors (D(2)R) in the striatum after hypoxia-ischemia in newborn rats. We show here an early and transient reduction of mRNA D(2)R in nonatrophic brains following hypoxia-ischemia. The left carotid artery of P7 rats was ligated followed by hypoxia for 2 h. The rats were sacrificed after 24 h, 48 h and 14 days. D(2)R mRNA was studied by in situ hybridization, the cell number by conventional histology, and neuronal and astrocyte differentiation by immunohistochemistry. A 20% reduction of striatal mRNA D(2)R occurred 24 h after hypoxia-ischemia, whereas no reduction was observed after 48 h and 14 days. There were no differences in total cell number and in the expression of neuronal (MAP-1, MAP-2) and astrocyte (GFAP) markers between both brain hemispheres nor between control and hypoxia-ischemia animals. The early decrease in mRNA D(2)R could explain the delayed reduced D(2)R after neonatal hypoxia-ischemia.

Neuroprotection of nerve growth factor-loaded microspheres on the D2 dopaminergic receptor positive-striatal neurones in quinolinic acid-lesioned rats: a quantitative autoradiographic assessment with iodobenzamide.

Huntington's disease (HD) results from the degeneration of striatal neurones, mainly gamma-aminobutyric acid (GABA)ergic projection neurones and lately cholinergic interneurones. The use of trophic factors as agents able to prevent such neural degeneration is a promising strategy. The aim of this study was to validate nerve growth factor-loaded (NGF-loaded) poly-D,L-lactide-co-glycolide (PLGA) microspheres for treatment of HD in a rat model with quinolinic acid lesion using autoradiographic study of D2 dopaminergic receptors (D2R). This target is expressed by about half of striatal neurones and its scintigraphic exploration has already been performed for the follow-up of this degenerative process. Ex vivo autoradiography of D2R performed with iodobenzamide, the widely used ligand for single photo emission computerized tomography, revealed slight neuroprotection. Moreover, tolerance of microspheres was demonstrated by in vitro autoradiography with the marker of gliosis, [(3)H]-PK 11195.

Modification of dopamine neurotransmission in the nucleus accumbens of rats deficient in n-3 polyunsaturated fatty acids.

We studied the effects of a diet chronically deficient in alpha-linolenic acid, the precursor of long-chain n-3 polyunsaturated fatty acids, on dopaminergic neurotransmission in the shell region of the nucleus accumbens of rats. In vivo microdialysis experiments showed increased basal levels of dopamine and decreased basal levels of metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in awake rats from the deficient group compared to controls. The release of dopamine under KCl stimulation was similar in both dietary groups. By contrast, the release of dopamine from the vesicular storage pool under tyramine stimulation was 90% lower in the deficient than in the control rats. Autoradiographic studies in the same cerebral region revealed a 60% reduction in the vesicular monoamine transporter sites in the deficient group. Dopamine D(2) receptors were 35% increased in these rats compared to controls, whereas no change occurred for D(1) receptors and membrane dopamine transporters. These results demonstrated that chronic n-3 polyunsaturated fatty acid deficiency modifies several factors of dopaminergic neurotransmission in the nucleus accumbens. These findings are in agreement with the changes in dopaminergic neurotransmission already observed in the frontal cortex, and with the behavioral disturbances described in these deficient rats.

Time course of changes in striatal dopamine transporters and D2 receptors with specific iodinated markers in a rat model of Parkinson's disease.

The time course of the loss in presynaptic dopamine transporters (DAT) and of the increase in postsynaptic dopamine D2 receptors (D2R) was studied in a rat model of Parkinson's disease. For this, in vitro autoradiographic experiments were performed in the striatum using (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methy lphenyl) nortropane (PE2I), a new single photon emission tomography (SPET) ligand for DAT, and iodobenzamide (IBZM), a SPET ligand for D2R. A significant decrease in [125I]PE2I binding was observed as early as 24 h after 6-hydroxydopamine lesion, whereas no change occurred in [125I]IBZM binding. At 48 h postlesion, PE2I binding was 50% decreased, while IBZM binding was 30% increased. Between 3 and 14 days postlesion, PE2I binding had almost totally disappeared and IBZM binding remained increased by around 40-50%. From these animal experiments, it can be assumed that PE2I would be very efficient for the detection of a reduction in the number of DAT reflecting neuronal loss, thus allowing early diagnosis of Parkinson's disease. The exploration of both DAT and D2R would improve follow-up of this disease.

A new iodinated tropane derivative (beta-CDIT) for in vivo dopamine transporter exploration: comparison with beta-CIT.

SPECT exploration of the dopamine transporter with tropane derivatives such as beta-CIT has already produced very valuable information in humans. However, the high affinity of this tracer for both dopamine and serotonin transporters and its slow in vivo kinetics provide the best images in humans more than 20 h after injection. In order to improve those properties, we performed structural changes in the tropane structure in the phenyl and nitrogen substituents for higher affinity and specificity and obtained a promising ligand, 2 beta-carbomethoxy-3 beta-(3',4' diclorophenyl)-8-(3-iodoprop-2E-enyl) nortropane (beta-CDIT). This iodinated ligand was characterized in vitro and in vivo in the rat in comparison with beta-CIT. In vitro competition studies revealed that beta-CIT and beta-CDIT similarly inhibited the binding of [3H]GBR 12935 (Ki = 27.5 and 29.0 nM, respectively). In contrast, competition studies with [3H]paroxetine and [3H]nisoxetine showed that beta-CDIT had a lower affinity for the serotonin transporter than beta-CIT (Ki = 50 and 3 nM, respectively) and also a lower affinity for the noradrenaline transporter than beta-CIT (Ki = 500 and 80 nM, respectively). In vivo studies in the rat showed that there was high and rapid uptake of [125I] beta-CDIT in the striatum. In addition, preinjection of GBR 12909 prevented accumulation of this ligand in the striatum by 80%, whereas only a 30% decrease was obtained for [125I] beta-CIT. It seems, therefore, that the combination of aromatic and nitrogen substitution improves the properties of tropane derivatives to provide an exclusive dopamine transporter ligand potentially usable in SPECT.

Iodinated PK 11195 as an ex vivo marker of neuronal injury in the lesioned rat brain.

In order to study the potentials of indirect and direct detection of neuronal damages in humans by single photon emission computerized tomography (SPECT), we compared ex vivo cerebral biodistribution of [125I]PK 11195 with that of [125I]TISCH in a rat model of unilateral striatal excitotoxic lesion. Experiments on in vitro binding with [3H]PK 11195 as a ligand for peripheral type benzodiazepine binding sites (PTBBS) and [3H]SCH 23390 as a ligand for dopamine D1 receptors were also performed to validate our experimental model. We observed a very high increase in the PTBBS and a dramatic decrease in the D1 receptors on the lesioned striatum compared to the intact side. Moreover, we showed that [125I]PK 11195 bound specifically to PTBBS in lesioned cerebral areas ex vivo 5 days after striatal infusion of quinolinic acid (600 nmoles). Increases of 192%, 168%, and 30% were obtained in the striatum, the cortex, and the hippocampus, respectively, on the lesioned side. These results showed that iodinated PK 11195 bound specifically to PTBBS ex vivo and that this binding was dramatically increased at the sites of brain lesion. This ligand could therefore be suitable to detect brain injuries in humans by SPECT, complementing the information given by ligands which image loss in a specific neuron population.

An iodinated derivative of moclobemide as potential radioligand for brain MAO-A exploration.

In vivo evaluation of MAO-A would be of great value for the diagnosis and follow-up of therapy of depression. In order to perform this exploration by SPECT, we developed an iodinated derivative of the reversible MAO-A inhibitor, moclobemide. Ro 11-9900 was synthesized and analysed by IR, NMR and HPLC. Radioiodination was carried out by nucleophilic exchange of [125I] on the brominated precursor, and yielded [125I]-Ro 11-9900 with high specific activity. In vitro experiments on rat brain homogenates showed that Ro 11-9900 had poor inhibitory activity on MAO-A, as already described for moclobemide. By contrast, in vivo biodistribution in the rat brain showed that [125I]-Ro 11-990 accumulated in a region corresponding to the localization of locus coeruleus known for its high density of MAO-A. Moreover, preinjection of the irreversible MAO-A inhibitor clorgyline (10 mg.kg-1) prevented accumulation of radioactivity by 40 to 60% and we found that the radioactivity in the brain corresponded exclusively to [125I]-Ro 11-9900 and not to a metabolite. [125I]-Ro 11-9900 was highly accumulated in the pineal gland, both on MAO-A and on MAO-B sites. We concluded that the unmetabolized iodinated derivative of moclobemide, Ro 11-9900, preferentially labeled MAO-A in vivo in the rat brain. This compound would therefore be a potential tracer for evaluation of MAO-A by SPECT.

Synthesis and characterization of [125I]N-(2-aminoethyl)-4-iodobenzamide as a selective monoamine oxidase B inhibitor.

We described the radiosynthesis of an analog of Ro 16-6491, [125I]N-(2-aminoethyl)-4-iodobenzamide, for SPECT exploration of the monoamine oxidase B (MAO-B) in human brain. The radiolabelling was carried out by nucleophilic exchange of the brominated precursor at solid-state phase in presence of ammonium sulphate. The radiochemical purity of radioiodinated product was higher than 95%. In comparison with Ro 16-6491, the in vitro studies showed a good selectivity of stable N-(2-aminoethyl)-4-iodobenzamide for MAO-B but a slightly lower affinity. Biodistribution studies in the rat showed a high and selective uptake of this compound in the pineal gland 1 h after i.v. injection. The cerebral uptake was low, but the coupling of [125I]N-(2-aminoethyl)-4-iodobenzamide with a lipophilic radical to enhance the passage through the blood-brain barrier can be envisaged.

[Quantification and monitoring of vascular resistance in the lower limbs by the Doppler method (animal model)]. / Quantification et monitorage des résistances vasculaires du membre inférieur. Par méthode doppler (modèle animal).

The object of this study was to define and validate a non-invasive method of evaluation and monitoring of vascular resistances in the leg. Blood flow velocity was measured by Doppler ultrasound in an animal model (ewe) with similar blood flow characteristics in the lower limb as man and allowing access to the required invasive measurements for validation of the method (pressure and flow). Vascular resistances distal to the measuring point (femoral, for example) were assessed using the resistance index R = D/S, S being the peak systolic deflection and D that of diastolic reflux of the Doppler spectral analysis of flow in the femoral artery. The values and variations of this resistance index were compared with the vascular resistances calculated from measurements of pressure and flow at the point of Doppler sampling and expressed in mmHg/ml/min. Femoral flow was measured by Doppler ultrasound (Doppler-echo), and mean pressure by an arterial catheter introduced into the abdominal aorta. Compression of the lower limb veins induced a venous return resulting in a reduction of cardiac output and femoral flow. During compression, femoral flow decreased by an average of 29% (p < 0.001) although mean pressure and heart rate did not change significantly. The femoral resistance index (Rf) increased by an average of 37.5% (p < 0.01) and vascular resistances increased by 45.9% (p < 0.01). Injection of 1 mg adrenaline induced peripheral vasoconstriction with an increase in blood pressure and a decrease in heart rate and femoral flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular resistance quantification in high flow resistance areas using the Doppler method.

The objective of the present study is to define and validate on an animal model (ewe) a new Doppler parameter for the assessment and monitoring of the vascular resistances in high resistance to flow areas (lower limbs, placenta with vascular disease). The high resistance index (HRI) was derived from the transmission line theory and defined as: HRI = D/S with S the amplitude of the systolic peak and D that of the diastolic reverse flow. Validation of the HRI was performed on adult ewes. Distal lower limb vascular resistances were evaluated from the Doppler femoral waveform (HRI) and compared with the classic vascular resistances (Rv), calculated from pressure and flow (mmHg/mL/min). The femoral flow variations were measured by duplex (echo-Doppler) method and the mean pressure through an arterial catheter inserted into the abdominal aorta. Two tests were used in this study: (1) A calibrated venous compression of the lower limbs extremity: The femoral flow dropped by 29%, the pressure and heart rate did not change, the HRI increased by 37% (p < 0.01), and the vascular resistance (Rv) by 46% (p < 0.01). (2) The intravenous injection of 1 mg adrenaline: The arterial pressure increased by 70% (p < 0.001), the heart rate and femoral flow dropped by 50% (p < 0.001), and 35% (p < 0.001), the HRI increased by 70% (p < 0.01) and the vascular resistances (Rv) by 140% (p < 0.01). During the two tests, the HRI changed in proportion with the "classic" vascular resistances.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the fetal PO2 changes by cerebral and umbilical Doppler on lamb fetuses during acute hypoxia.

The objective of the present study was to validate one or a combination of fetal Doppler parameters in order to assess acute fetal hypoxia in an ovine model. Acute hypoxia was induced by reducing umbilical, or maternal aortic flow (approx. 70%). A CW Doppler probe was fixed on the fetal cervical skin, facing the internal carotid artery and the fetal abdominal skin adjacent to the umbilical arteries. (The angle between Doppler beam and flow vector remained constant.) A "Doptek 3000" spectrum analyser was used to measure the maximal and mean Doppler frequencies. Heart rate (HR), umbilical blood flow (UBF), carotid blood flow (CBF), umbilical RI (URI), cerebral RI (CRI) and cerebroplacental ratio (CPR = CRI/URI) were calculated in real time. A catheter was inserted into the fetal femoral artery, for blood gas (PO2, PCO2 pH) and blood pressure (BP) measurements. After 1 min of aorta compression (70% aortic flow reduction), the URI increased by 10% (P < 0.05), and the UBF decreased by 10% (P < 0.05), but the CRI decreased by 20% (P < 0.02), and the CBF did not change significantly. Fetal PO2 and CPR fell down after 1 min (59% and 38%, respectively; P < 0.001), although strong fetal heart rate decelerations were observed. The blood pressure, PCO2 and pH did not change significantly during this test. Throughout the 12 min of cord compression (70% umbilical flow reduction) the URI increased (70% to 80% P < 0.001), and the UBF decreased (approx. 60%; P < 0.001), but the CRI decreased (approx. 25%; P < 0.01), and the CBF remained constant (+/- 5%; ns). Fetal PO2 and CPR all decreased during the compression (30% to 44% and 40% to 60%, respectively; P < 0.001). HR, pH and PCO2 did not change significantly. During cord compression the blood pressure did not change significantly. In both cases, the CPR decreased significantly (P < 0.001) with the PO2 in the same direction and with a comparable amplitude (-30% to -50%). Nevertheless, the drop in CPR was greater during cord compression than during aorta compression, probably because the compression of the cord induced a central hypovolemia in addition to the hypoxia. The CPR was found to be the hemodynamic parameter that followed most closely the PO2 acute changes. The amplitude of the variations of this parameter (-30% to -50%) were quite similar to those of the PO2 during the period of acute hypoxia.(ABSTRACT TRUNCATED AT 400 WORDS)

Concurrent changes in intracranial pressure, cerebral blood flow velocity, and brain energy metabolism in rabbits with acute intracranial hypertension.

The relationship between intracranial pressure or cerebral perfusion pressure (CPP), cerebral blood flow, and brain energy failure is unpredictable throughout the development of acute intracranial hypertension. The purpose of the present study was to correlate intracranial pressure with cerebral blood flow velocities and brain energy metabolism in adult rabbits. The acute intracranial hypertension was achieved by pressure transmission. Transcranial Doppler wave-forms were obtained from the basilar artery for monitoring cerebral blood flow velocities. 31P-Magnetic resonance spectroscopy was used to assess brain energy metabolism. The diastolic blood flow velocity began to decrease significantly (34.5%) when the intracranial pressure was equal to half the diastolic arterial pressure for a CPP of 36 +/- 18 mmHg. Circulatory cerebral resistances increased significantly (55%) for the same value of CPP. Diastolic frequency was near zero when intracranial pressure approached diastolic arterial pressure (51 +/- 12 mmHg), corresponding to a CPP of 30 +/- 15 mmHg. At the same time, only a tendency for brain energy metabolism to decrease was observed. Consequently, transcranial Doppler sonography could be proposed for the follow-up of intracranial hypertension. Magnetic resonance spectroscopy could help to monitor these patients and could be especially proposed in case of high intracranial pressure (near diastolic arterial pressure). The joint use of these two methods would help in making appropriate therapeutic decision in humans.

New implanted Doppler sensors for the assessment of the main fetal hemodynamics.

If the fetal Doppler examination during human pregnancies provides useful information to the obstetrician, it does not allow us to collect all the biological and haemodynamic data required to understand the physiopathological mechanisms involved in the development of intrauterine growth retardation (IUGR) and hypoxia. With the animal model, it is possible to have access to the blood pressure, the blood velocity and volume, to collect blood sampling and to perform pharmacological tests, or to simulate some human pathology. Several studies have been already carried out on lamb fetuses using electromagnetic flowmeters placed around the cord and catheters, with pressure sensors placed inside the fetal aorta. Most of the time only the umbilical flow was assessed on the fetal side. The aim of the present work was to develop a new Doppler system able to monitor in real time and simultaneously, the fetal cerebral and the umbilical arterial flows as well as the uterine circulation. New flat Doppler probes have been designed, to be implanted on the fetus and on the mother, which makes possible the atraumatic assessment of fetal and maternal flows during approximately 20 days. The 4-MHz CW Doppler probe consists of two rectangular piezoelectric transducers of 13-mm wide, preoriented at 45 degrees from the surface of the probe, placed in a 6-mm-high plastic case in which small holes are made to sew the probe on the fetal skin. The sensors are fixed on the fetal skin, facing the umbilical cord, the fetal cerebral arteries and in front of the uterine arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of cerebral blood flow in rabbits with transcranial Doppler sonography: first results.

A transcranial Doppler sonographic system with a special 2-MHz probe for prolonged experimentation has been developed for Doppler waveform recordings from the basilar artery on rabbits. Measurements were made from the maximum velocity wave form, and Pourcelot's resistance index was used to express the results. The diastolic flow, similar to that observed in humans in physiological conditions, decreases with increased intracranial pressure during chronic intracranial hypertension. This Doppler model may be valuable for assessing rapid changes of cerebral blood flow in conscious animals during prolonged or acute experimental procedures.