Efficient gene expression from integration-deficient lentiviral vectors in the spinal cord.

Gene transfer to spinal cord cells may be crucial for therapy in spinal muscular atrophy, amyotrophic lateral sclerosis and spinal cord injury. Lentiviral vectors are efficient for transduction of a variety of cells, but like all integrating vectors they pose a risk of insertional mutagenesis. Integration-deficient lentiviral vectors (IDLVs) remain episomal but retain the transduction efficiency of standard integrating lentiviral vectors, particularly when the episomes are not diluted out through repeated cell division. We have now applied IDLVs for transduction of spinal cord in vitro, in explants and in vivo. Our results demonstrate similar efficiency of eGFP expression from integrating lentiviral vectors and IDLVs in most cell types analyzed, including motor neurons, interneurons, dorsal root ganglia (DRG) neurons and astroglia. IDLV-mediated expression of pro-glial-cell-derived neurotrophic factor (Gdnf) rescues motor neuron cultures from death caused by removal of exogenous trophic support. IDLVs also mediate efficient RNA interference in DRG neuron cultures. After intraparenchymal injection in the rat and mouse cervical and lumbar regions in vivo, transduction is mainly neuronal, with both motor neurons and interneurons being efficiently targeted. These results suggest that IDLVs could be efficient and safer tools for spinal cord transduction in future therapeutic strategies.

A reduction in milking frequency and feed allowance improves dairy cow immune status.

Twice-daily milking is the most common milking regimen used globally. A reduction in milking frequency to once daily, combined with a reduced feed allowance (FA), could reduce the physiological stress associated with the transition to peak milk production, and hence improve immune function. This study investigated how milking frequency and FA affect dairy cow immune status. Cows (n = 48) were milked once a day (OAD) or twice a day (TAD) on 1 of 2 FA: high (HFA) or low (LFA), in a 2 × 2 factorial arrangement of treatments. After the mean calving date of March 11, HFA cows were offered ad libitum grass silage and 7 kg of concentrates/cow per day until March 22, then 4 kg of concentrates/cow per day until April 17, and thereafter allocated 31.3 kg of dry matter (DM) grass/cow per day. The LFA cows were offered 4 kg of concentrates/cow per day, 1 kg of concentrates/cow per day, and allocated 19 kg of DM grass/cow per day for the same respective periods. Milk yield was recorded daily and body condition score weekly, and somatic cell count was performed at approximately 2-wk intervals. Blood samples were collected prepartum (d -7 to -1) and at d 1 to 7, d 14 to 21, and d 42 to 49 postpartum. Total and differential leukocyte percentage, IFN-γ production in response to concanavalin A and phytohemagglutinin, and cortisol, haptoglobin (Hp), and serum amyloid A (SAA) concentrations were evaluated. Cows milked OAD had reduced milk yield and body reserve mobilization, but higher somatic cell counts. Milking frequency and diet had no effect on total leukocyte counts. Cows milked OAD had a higher lymphocyte percentage and lower monocyte percentage, and tended to have a lower neutrophil percentage than cows milked TAD. In addition, the LFA cows had a higher eosinophil percentage than cows fed the HFA. Milking frequency and diet had no effect on IFN-γ, Hp, SAA, or cortisol production. Utilization of strategies to reduce milk yield at the beginning of the lactation could not only reduce body reserve mobilization, but also help to maintain a functioning immune system, and thus improve cow welfare.

My pigs are ok, why change? - animal welfare accounts of pig farmers.

Intensive pig production systems are a source of stress, which is linked to reduced animal welfare and increased antimicrobial use. As the gatekeepers of the welfare of the animals under their care, farmers are seen as the stakeholder responsible for improving animal welfare. The aim of this study was to explore the knowledge and attitudes of pig farmers towards pig welfare and the impact of such attitudes on farmers' selection of management strategies on the farm. We conducted in-depth semi-structured interviews with 44 pig farmers in one of the main pig producing regions of Brazil. Interviews covered knowledge and attitudes towards pig sentience and behaviour and welfare-related issues commonly observed in intensive pig farms (belly-nosing, fights, tail-biting, diarrhoea and castration without pain control) and farmers' conception and attitudes towards pig welfare. We identified many management and animal-based indicators of poor welfare, such as the use of painful and stressful management practices and use of environments that limit the expression of natural behaviours. However, most farmers were satisfied with animal welfare standards at their farms. Farmers' perceptions are aligned with their understanding of animal welfare. Although they identified all the dimensions that impact the welfare of a pig on a farm (affect, biological functioning and naturalness), their social reality, industry demands and available advice pushed them to perceive their range of action limited to biological and environmental aspects of the animals that do not necessarily benefit affective state. This precluded farmers from making associations between good health and the animal's ability to express a full behavioural repertoire, as well as from viewing abnormal behaviours as problems. The negative consequences for the welfare of the animals were commonly alleviated by routines that relied on constant use of medication, including high dependence on antibiotics. Expressions of estrangement from the production chain were common voices among the participants. This suggests that farmers may not be sufficiently informed or engaged in responding to consumers' expectations and commitments made by companies, which can pose a severe economic risk for farmers. The findings of this study indicate that economic, technical and social factors restrict farmers' autonomy and their ability to perform their role as stewards of animal welfare. (Re)connecting different human, animal and environmental interests may be a step to changing this scenario.

Dairy calves' mortality survey and associated management practices in smallholding, pasture-based herds in southern Brazil.

Our retrospective, cross-sectional study aimed to identify the mortality rate of preweaned heifer calves and associated herd-management factors in family-run dairy holdings in the state of Rio Grande do Sul, Brazil. A survey was made available through an online platform to all the municipalities (n = 494) with a state official extension office (Emater), allowing for any milk producer in RS raising the calves on-farm to take part in the study. A total of 1451 farmers responded to the survey between October 2014 and December 2015. Yearly total mortality, stillbirths and post-natal mortality to weaning were calculated from responses. The association of herd characteristics and practices with herd mortality was evaluated using multilevel multivariable logistic regression models. Herd mortality was modelled as a two vector binded variable that included the number of failures (n = female calves born alive but dead before weaning) and successes (n = weaned female calves) on-farm in the past 12 months. Models included region as random effect. The herds had 13 lactating cows (median, range 1-130). Pure breed herds, especially Holstein, predominated (81 %). Milk production was 190 L/d (median, range 7-4000). Total mortality rate was 8.5 % (1065/12563, median 0.0 %, Q1 - Q3 0.0-14.3 %/farm), and stillbirths 1.7 % (207/12563; median 0.0 %, Q1 - Q3 0.0 - 0 %/farm). Post-natal mortality rate was 6.9 % (858/12356, median 0.0 %, Q1 - Q3 0.0-11.1 %/farm). In 89.2 % of the farms, no stillborn calf was reported, and in only 2.8 % there was more than one case. In 67 % of farms, no death calf born alive was reported; in 33 %, an average of 2 deaths/farm (range 1-12 deaths/farm) was reported. In 16 % of the farms, mortality was ≥ 20 %, and in 4.3 % it was ≥ 50 %. The main reported causes of death were diarrhoea and unknown causes. Higher mortality was associated with herds with mixed breeds (OR = 1.3, CI = 1.09-1.59), performing unattended calvings (OR = 1.2; IC = 1.04-1.40), leaving the calf for long periods with the cow (OR = 1.21, CI = 1.00-1.45), and housing various calves in a pen (OR = 1.4, CI = 1.20-1.60). Furthermore, reduced mortality was associated with medium size herds (i.e. 21-40 lactating cows; OR = 0.82, CI = 0.69-0.97). Although the mortality rate observed is conservatively lower or equal to other international reports, it is higher than could be desired. The factors identified as associated to mortality are understood as proxies for the poor quality of management of the practices adopted. Thus, reduction of mortality is at hand without representing major infrastructural changes.

Vascular endothelial growth factor protects motoneurons from serum deprivation-induced cell death through phosphatidylinositol 3-kinase-mediated p38 mitogen-activated protein kinase inhibition.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective degeneration and death of motoneurons in the spinal cord, brainstem and motor cortex which causes progressive muscle weakness and paralysis. Although the molecular mechanisms causing the disease remain unknown, excitotoxicity and loss of trophic support have been proposed as causes of degeneration. The present study was designed to elucidate the mechanisms of motoneuron death induced by serum deprivation and the potential neuroprotective effects of vascular endothelial growth factor (VEGF) in dissociated and organotypic rat spinal cord cultures. Serum withdrawal induced apoptotic cell death in dissociated spinal cord cultures, which was prevented in the presence of VEGF. In organotypic spinal cord cultures, low serum-induced motoneuron death was mediated by the stress-related kinase p38 mitogen-activated protein kinase (p38MAPK), as it was reversed by the p38MAPK inhibitor SB203580. In these cultures, exposure to VEGF blocked p38MAPK phosphorylation and prevented the demise of motoneurons. These effects of VEGF were mediated through the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) signal transduction pathway, as they were blocked in the presence of the PI3-K inhibitor LY294002. In addition, serum deprivation induced down-regulation of the anti-apoptotic protein Bcl-2 and this effect was prevented both by SB203580 and by VEGF via the PI3-K/Akt pathway. Therefore, Bcl-2 could also play an important role in the neuroprotection induced by VEGF in spinal cord cultures. Together, these findings indicate that VEGF prevents motoneuron death induced by serum deprivation blocking the activity of p38MAPK via the PI3-K/Akt signaling pathway.

Transplacental exchange of moxidectin after maternal or fetal intravenous administration in sheep.

The transplacental exchange of moxidectin after maternal or fetal intravenous (i.v.) administration was studied using the chronically catheterized fetal sheep model. Nine pregnant Suffolk Down sheep of 65.7 +/- 5.9 kg body weight (bw) were surgically prepared to insert polyvinyl catheters in the fetal femoral artery and vein and amniotic sac. The ewes were randomly assigned to two experimental groups. In group 1 (maternal injection) five ewes were treated with an i.v. bolus of 0.2 mg of moxidectin/kg bw. In group 2, (fetal injection) an i.v. bolus of 1 mg of moxidectin was administered to the four fetuses by femoral vein catheters. Maternal and fetal blood and amniotic fluid samples were taken before and after moxidectin administration for a 144 h post-treatment period. Samples were analyzed by liquid chromatography. A noncompartmental pharmacokinetic analysis was performed and statistical differences were determined by mean of parametric and nonparametric statistical tests. Pharmacokinetic differences observed in maternal variables were shorter elimination half-life and mean residence time compared with values previously reported for ivermectin. Drug diffusion from maternal to fetal circulation (AUC(0-t) = 232.6 +/- 72.5 ng.h/mL) was statistically not different (P = 0.09) compared with fetal to maternal diffusion (AUC(0-t) = 158.0 +/- 21.6 ng.h/mL). Fetuses showed significantly (P = 0.008) lower drug body clearance values compared with those observed in the maternal side. Considering the observed transplacental passages between materno-fetal or feto-maternal circulations, we conclude that the placental barrier is not effective in preventing the moxidectin diffusion between mother and fetus.

Nuevo coronavirus (COVID-19) en población general y pediátrica: una revisión epidemiológica: Chile 2020 / Novel coronavirus (COVID-19) in general and pediatric population: an epidemiological review: Chile 2020

Since December 2019, the outbreak of a novel coronavirus SARS Cov-2 has been reported in Wuhan, China. Currently, the new coronavirus disease has been declared a worldwide pandemic. Compared to adults, reporting of cases in pediatric patients has been significant smaller. The objective of this article is to provide epidemiological information of COVID-19, especially pediatric. Most of the confirmed cases of children are declared to be a cluster disease. The clinic is oligosymptomatic, less severe and with concentrated risk in children under 1 year of age and with comorbidity. In Chile, pediatric patients represent about 6% of the total number of infected and overall lethality is significantly lower than adults. The main control measures to reduce effective reproduction are mass testing, social distancing and school closure, without dismissing individual responsibility. The adequate supply of personal protection elements is key to avoid nosocomial infection and the compromise of healthcare providers.

A partir de diciembre del 2019, se ha reportado el brote de una nueva infección por SARS Cov-2 en Wuhan, China. Actualmente, la enfermedad por el nuevo coronavirus 2019 ha alcanzado el estatus de pandemia. El reporte de casos en pacientes pediátricos ha sido escaso. El objetivo de este artículo es entregar información epidemiológica del COVID-19, especialmente pediátrica. Los niños han presentado enfermedad en clusters, secundaria a contacto con parientes enfermos. La clínica es oligosintomática, menos severa y mayor riesgo concentrado en menores de 1 año y con comorbilidad. En Chile, los pacientes pediátricos representan cerca del 6% del total y la letalidad global es notablemente más baja que en adultos. Las principales medidas de control para la reducción de la reproducción efectiva son el testeo masivo, distanciamiento social y cierre escolar, sin desestimar la responsabilidad individual. El adecuado abastecimiento de elementos de protección personal es clave para evitar la infección nosocomial y del personal de salud.

Mu-opioid receptor activation prevents apoptosis following serum withdrawal in differentiated SH-SY5Y cells and cortical neurons via phosphatidylinositol 3-kinase.

Opioid peptides and alkaloids exert their effects via G protein-coupled receptors (GPCRs). It has been shown that, in addition to trophic factors, some GPCRs are able to activate the phosphatidylinositol 3-kinase/Akt (PI 3-K/Akt) signal transduction pathway, thus leading to cell survival. The aim of this study was to test whether activation of mu-opioid receptors has protective effects on serum withdrawal-induced cell death and to study the possible implication of PI 3-K in this process. In SH-SY5Y neuroblastoma cells fully differentiated by exposure to retinoic acid for five days, the enkephalin derivative selective mu-agonist DAMGO (0.1-2 microM) and the alkaloid morphine (0.1-10 microM) promoted cell survival after serum deprivation (MTT and trypan blue exclusion assays), without inducing cell proliferation. These effects were fully reversed by naloxone, by the selective mu-antagonist beta-funaltrexamine (beta-FNA) and also by the specific PI 3-K inhibitor LY294002. The two agonists stimulated Akt phosphorylation and the effect was also abolished by beta-FNA and by LY294002. In mouse primary cortical neurons, DAMGO reduced the percentage of apoptosis after 6, 12, 24 and 48 h of serum withdrawal; as determined by Hoechst staining. This effect was blocked by beta-FNA, by pre-treatment with pertussis toxin and by LY294002. DAMGO also stimulated Akt phosphorylation via PI 3-K in this primary neuronal culture. Together, these results indicate that stimulation of the mu-opioid receptor promotes neuronal survival in a G(i/o)-linked, PI 3-K-dependent signaling cascade and suggest that Akt may be a key downstream kinase involved in this anti-apoptotic effect.

Synaptic remodeling in the rat arcuate nucleus during the estrous cycle.

Adult female rats showing regular vaginal cycles were studied in order to determine the number of axosomatic synapses in thin sections of the arcuate nucleus. The number of synapses per length of perikaryal membrane was significantly decreased in estrus, compared to other days of the estrous cycle (P less than 0.05). The reduction in the number of synapses in estrus was accompanied by a decrease in the percentage of the average length of perikaryal membrane covered by presynaptic terminals and by an increase in the percentage of membrane in close apposition of glial processes. Since the average perikaryal perimeter was not significantly changed during the estrous cycle, these results indicate a net decrease in the number of arcuate nucleus axosomatic synapses between proestrus and estrus, with a reinnervation of arcuate neurons between estrus and metestrus. These results suggest that there is a physiological synaptic turnover in the arcuate nucleus of the rat during the estrous cycle.

The effects of phenelzine and other monoamine oxidase inhibitor antidepressants on brain and liver I2 imidazoline-preferring receptors.

1. The binding of [3H]-idazoxan in the presence of 10(-6) M (-)-adrenaline was used to quantitate I2 imidazoline-preferring receptors in the rat brain and liver after chronic treatment with various irreversible and reversible monoamine oxidase (MAO) inhibitors. 2. Chronic treatment (7-14 days) with the irreversible MAO inhibitors, phenelzine (1-20 mg kg-1, i.p.), isocarboxazid (10 mg kg-1, i.p.), clorgyline (3 mg kg-1, i.p.) and tranylcypromine (10 mg kg-1, i.p.) markedly decreased (21-71%) the density of I2 imidazoline-preferring receptors in the rat brain and liver. In contrast, chronic treatment (7 days) with the reversible MAO-A inhibitors, moclobemide (1 and 10 mg kg-1, i.p.) or chlordimeform (10 mg kg-1, i.p.) or with the reversible MAO-B inhibitor Ro 16-6491 (1 and 10 mg kg-1, i.p.) did not alter the density of I2 imidazoline-preferring receptors in the rat brain and liver; except for the higher dose of Ro 16-6491 which only decreased the density of these putative receptors in the liver (38%). 3. In vitro, phenelzine, clorgyline, 3-phenylpropargylamine, tranylcypromine and chlordimeform displaced the binding of [3H]-idazoxan to brain and liver I2 imidazoline-preferring receptors from two distinct binding sites. Phenelzine, 3-phenylpropargylamine and tranylcypromine displayed moderate affinity (KiH = 0.3-6 microM) for brain and liver I2 imidazoline-preferring receptors; whereas chlordimeform displayed high affinity (KiH = 6 nM) for these receptors in the two tissues studied, Clorgyline displayed very high affinity for rat brain (KiH = 40 pM) but not for rat liver I2 imidazoline-preferring receptors (KiH = 169 nM). 4. Preincubation of cortical or liver membranes with phenelzine (10-4 M for 30 min) did not alter the total density of I2 imidazoline-preferring receptors, indicating that this irreversible MAO inhibitor does not irreversibly bind to I2 imidazoline-preferring receptors. In contrast, preincubation with 10-6 Mclorgyline reduced by 40% the Bmax of [3H]-idazoxan to brain and liver I2 imidazoline-preferring receptors.5. Chronic treatment (7 days) with the inducers of cytochrome P-450 enzymes phenobarbitone (40 or 80 mg kg-1, i.p.), 3-methylcholanthrene (20 mg kg-1, i.p.) or 2-methylimidazole (40 mg kg-1, i.p.) did not alter the binding parameters of [3H]-idazoxan to brain and liver 12 imidazoline-preferring receptors.The compound SKF 525A, a potent inhibitor of cytochrome P-450 enzymes which forms a tight but reversible complex with the haemoprotein, completely displaced with moderate affinity (KiH = 2-10 microM)the specific binding of [3H]-idazoxan to brain and liver 12 imidazoline-preferring receptors. Preincubation of total liver homogenates with 3 x 10-4 M phenelzine in the presence of 10-3 M NADH, a treatment that irreversibly inactivates the haeme group of cytochrome P-450, did not reduce the density of liver I2 imidazoline-preferring receptors. These results discounted a possible interaction of [3H]-idazoxan with the haeme group of cytochrome P-450 enzymes.6. Together the results indicate that the down-regulation of I2 imidazoline-preferring receptors is associated with an irreversible inactivation of MAO (at least in the brain) that is not related either to the affinity of the MAO inhibitors for I2 imidazoline-preferring receptors or to an irreversible binding to these putative receptors. These findings indicate a novel effect of irreversible MAO inhibitors in the brain and suggest a new target for these compounds that could be of relevance in the treatment of depression, a disease in which an increased density of brain I2 imidazoline-preferring receptors has been reported.

The effects of chronic imidazoline drug treatment on glial fibrillary acidic protein concentrations in rat brain.

1. The concentration of the astrocytic marker, glial fibrillary acidic protein (GFAP) was quantitated by immunoblotting (western blotting) in the rat brain after treatment with various imidazoline drugs and other agents. 2. Chronic (7 days) but not acute (1 day) treatment with the imidazoline drugs, cirazoline (1 mg kg-1, i.p.) and idazoxan (10 mg kg-1, i.p.), but not with the structurally related alpha 2-adrenoceptor antagonists, RX821002 (2-methoxy idazoxan) (10 mg kg-1, i.p.) and efaroxan (10 mg kg-1, i.p.), markedly increased (45%) GFAP immunoreactivity in the rat cerebral cortex. Chronic treatment (7 days) with yohimbine (10 mg kg-1, i.p.), a non-imidazoline alpha 2-adrenoceptor antagonist, did not significantly modify GFAP immunoreactivity in the cerebral cortex. 3. Chronic treatment (7 days) with cirazoline and idazoxan did not alter the density of brain monoamine oxidase (MAO)-B sites labelled by [3H]-Ro 19-6327 (lazabemide), another relevant astroglial marker. Moreover, these imidazoline drug treatments did not modify the levels of alpha-tubulin in the cerebral cortex. These negative results reinforced the specificity of the effects of imidazoline drugs on GFAP. 4. Irreversible inactivation of brain alpha 2-adrenoceptors (and other neurotransmitters receptors) after treatment with an optimal dose of the peptide-coupling agent EEDQ (1.6 mg kg-1, i.p., for 6-24 h) did not alter GFAP immunoreactivity in the cerebral cortex. These results further disproved the involvement of these receptors on astroglial cells in the tonic control of GFAP levels.5. The binding of [3H]-idazoxan in the presence of 10-6 M (-)-adrenaline was used to quantitate in parallel 12-imidazoline preferring sites in the rat brain after the same treatments. Chronic treatment (7 days) with cirazoline and idazoxan, but not with RX821002, efaroxan or yohimbine, significantly increased (25%) the density of I2-sites in the cerebral cortex. The up-regulation of I2-sites induced by cirazoline was not observed in the liver, a tissue that also expresses 12-sites but lacks glial cells.6. A strong correlation (r = 0.97) was found when the mean percentage changes in GFAP immuno reactivity were related to the mean percentage changes in 12 imidazoline sites after the various drug treatments.7. Together the results suggest a direct physiological function of glial I2-imidazoline preferring sites in the regulation of GFAP levels.

Attenuation of tolerance to opioid-induced antinociception and protection against morphine-induced decrease of neurofilament proteins by idazoxan and other I2-imidazoline ligands.

1. Agmatine, the proposed endogenous ligand for imidazoline receptors, has been shown to attenuate tolerance to morphine-induced antinociception (Kolesnikov el al., 1996). The main aim of this study was to assess if idazoxan, an alpha2-adrenoceptor antagonist that also interacts with imidazoline receptors, could also modulate opioid tolerance in rats and to establish which type of imidazoline receptors (or other receptors) are involved. 2. Antinociceptive responses to opioid drugs were determined by the tail-flick test. The acute administration of morphine (10 mg kg(-1), i.p., 30 min) or pentazocine (10 mg kg(-1), i.p., 30 min) resulted in marked increases in tail-flick latencies (TFLs). As expected, the initial antinociceptive response to the opiates was lost after chronic (13 days) treatment (tolerance). When idazoxan (10 mg kg(-1), i.p.) was given chronically 30 min before the opiates it completely prevented morphine tolerance and markedly attenuated tolerance to pentazocine (TFLs increased by 71-143% at day 13). Idazoxan alone did not modify TFLs. 3. The concurrent chronic administration (10 mg kg(-1), i.p., 13 days) of 2-BFI, LSL 60101, and LSL 61122 (valldemossine), selective and potent I2-imidazoline receptor ligands, and morphine (10 mg kg(-1), i.p.), also prevented or attenuated morphine tolerance (TFLs increased by 64 172% at day 13). This attenuation of morphine tolerance was still apparent six days after discontinuation of the chronic treatment with LSL 60101-morphine. The acute treatment with these drugs did not potentiate morphine-induced antinociception. These drugs alone did not modify TFLs. Together, these results indicated the specific involvement of I2-imidazoline receptors in the modulation of opioid tolerance. 4. The concurrent chronic (13 days) administration of RX821002 (10 mg kg(-1), i.p.) and RS-15385-197 (1 mg kg(-1), i.p.), selective alpha2-adrenoceptor antagonists, and morphine (10 mg kg(-1), i.p.), did not attenuate morphine tolerance. Similarly, the concurrent chronic treatment of moxonidine (1 mg kg(-1), i.p.), a mixed I(1)-imidazoline receptor and alpha2-adrenoceptor agonist, and morphine (10 mg kg(-1), i.p.), did not alter the development of tolerance to the opiate. These results discounted the involvement of alpha2-adrenoceptors and I(1)-imidazoline receptors in the modulatory effect of idazoxan on opioid tolerance. 5. Idazoxan and other imidazol(ine) drugs fully inhibited [3H]-(+)-MK-801 binding to N-methyl-D-aspartate (NMDA) receptors in the rat cerebral cortex with low potencies (Ki: 37-190 microM). The potencies of the imidazolines idazoxan, RX821002 and moxonidine were similar, indicating a lack of relationship between potency on NMDA receptors and ability to attenuate opioid tolerance. These results suggested that modulation of opioid tolerance by idazoxan is not related to NMDA receptors blockade. 6. Chronic treatment (13 days) with morphine (10 mg kg(-1), i.p.) was associated with a marked decrease (49%) in immunolabelled neurofilament proteins (NF-L) in the frontal cortex of morphine-tolerant rats, suggesting the induction of neuronal damage. Chronic treatment (13 days) with idazoxan (10 mg kg(-1)) and LSL 60101 (10 mg kg(-1)) did not modify the levels of NF-L proteins in brain. Interestingly, the concurrent chronic treatment (13 days) of idazoxan or LSL 60101 and morphine, completely reversed the morphine-induced decrease in NF-L immunoreactivity, suggesting a neuroprotective role for these drugs. 7. Together, the results indicate that chronic treatment with I2-imidazoline ligands attenuates the development of tolerance to opiate drugs and may induce neuroprotective effects on chronic opiate treatment. Moreover, these findings offer the I2-imidazoline ligands as promising therapeutic coadjuvants in the management of chronic pain with opiate drugs.

Induction of reactive astrocytosis and prevention of motoneuron cell death by the I(2)-imidazoline receptor ligand LSL 60101.

I(2)-imidazoline receptors are mainly expressed on glial cells in the rat brain. This study was designed to test the effect of treatment with the I(2)-imidazoline selective receptor ligand LSL 60101 [2-(2-benzofuranyl)imidazole] on the morphology of astrocytes in the neonate and adult rat brain, and to explore the putative neuroprotective effects of this glial response. Short-term (3 days) or chronic (7-10 days) treatment with LSL 60101 (1 mg kg(-1), i.p. every 12 h) enhanced the area covered by astroglial cells in sections of facial motor nucleus from neonate rats processed for glial fibrillary acidic protein (GFAP) immunostaining. Facial motoneurons surrounded by positive glial cell processes were frequently observed in sections of LSL 60101-treated rats. A similar glial response was observed in the parietal cortex of adult rats after chronic (10 days) treatment with LSL 60101 (10 mg kg(-1), i.p. every 12 h). Western-blot detection of the specific astroglial glutamate transporter GLT-1, indicated increased immunoreactivity after LSL 60101 treatment in the pons of neonate and in the parietoccipital cortex of adult rats. In the facial motor nucleus of neonate rats, the glial response after LSL 60101 treatment was associated to a redistribution of the immunofluorescence of the basic fibroblast growth factor (FGF-2) from the perinuclear area of motoneurons to cover most of their cytoplasm, suggesting a translocation of this mitogenic and neurotrophic factor towards secretion pathways. The neuroprotective potential of the above effects of LSL 60101 treatment was tested after neonatal axotomy of facial motor nucleus. Treatment with LSL 60101 (1 mg kg(-1), i.p. every 12 h from day 0 to day 10 after birth) significantly reduced (38%) motoneuron death rate 7 days after facial nerve axotomy performed on day 3 after birth. It is concluded that treatment with the I(2)-imidazoline selective receptor ligand LSL 60101 provokes morphological/biochemical changes in astroglia that are neuroprotective after neonatal axotomy.

Chronic treatment with the monoamine oxidase inhibitors clorgyline and pargyline down-regulates non-adrenoceptor [3H]-idazoxan binding sites in the rat brain.

1. The binding of [3H]-idazoxan in the presence of 10(-6) M (-)-adrenaline was used to quantitate non-adrenoceptor idazoxan binding sites (NAIBS) in the rat brain after treatment with various psychotropic drugs. 2. Chronic treatment (14 days) with the monoamine oxidase (MAO) inhibitors clorgyline (0.3-10 mg kg-1, i.p.) and pargyline (10 mg kg-1, i.p.), but not with Ro 41-1049 (1 mg kg-1, i.p.), markedly decreased (30-50%) the density of NAIBS in the cerebral cortex without any apparent change in the affinity of the radioligand. 3. Acute (1 day) and/or chronic treatments (14 days) with other psychotropic drugs such as desipramine (3 mg kg-1, i.p.), cocaine (10 mg kg-1, i.p.), reserpine (0.12 mg kg-1, s.c.), haloperidol (1 mg kg-1, i.p.) and diazepam (10 mg kg-1, i.p.) did not alter the density of NAIBS in the cerebral cortex. 4. In vitro, the propargylamines clorgyline, pargyline and deprenyl displaced the binding of [3H]-idazoxan to NAIBS from two distinct sites, but only clorgyline displayed an apparent very high affinity for a relevant population of NAIBS (KiH = 40 pM; KiL = 10.6 microM). The structurally diverse MAO inhibitors Ro 16-6491 (selective for MAO-B) and Ro 41-1049 (selective for MAO-A), as well as the other psychotropic drugs (desipramine, cocaine, reserpine and haloperidol) displaced the binding of [3H]-idazoxan to NAIBS monophasically and with very low potencies. As expected, the MAO inhibitors clorgyline and Ro 41-1049 displaced the binding of [3H]-Ro 41-1049 to MAO-A monophasically and with high potencies (Ki values: 0.18 nM and 22 nM, respectively). In contrast, idazoxan displayed very low affinity (Ki =40 microM) against the binding of pH]-Ro 41-1049 to MAO-A. These results disprove a direct interaction between [3H]-idazoxan and the enzyme MAO.5. Preincubation of cortical membranes with clorgyline (10-9M or 10-6 M for 30 min) or pargyline(10-6 M or 10-5M for 30 min), reduced by 30-50% and by 17-30%, respectively, the total density of NAIBS without any apparent change in the affinity of the radioligand. Preincubation with 10-6M clorgyline did not alter the affinity of cirazoline for the two populations of NAIBS, but reduced by 60%the binding of [3H]-idazoxan to the high affinity site without affecting the binding of the radioligand to the low affinity site. These results indicate that the two MAO inhibitors irreversibly block the binding of[3H]-idazoxan to NAIBS.6. In vivo, however, various acute treatments with clorgyline (1-20 mg kg-1, i.p.) for different time intervals (6-48 h) did not alter the density of NAIBS. In vivo, only very high doses of clorgyline (40 and 80 mg kg-1, i.p.) induced modest decreases (21-28%) in the density of NAIBS in the cerebral cortex.7. Together the results indicate that the irreversible binding of clorgyline and pargyline to NAIBSfound in vitro does not fully explain the marked decreases in the density of NAIBS found in vivo after the chronic treatments. It is suggested that the down-regulation of NAIBS induced in vivo by clorgyline and pargyline, through a direct or indirect mechanism, may have functional implications.

Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat liver.

1. I2-Imidazoline sites ([3H]-idazoxan binding) have been identified on monoamine oxidase (MAO) and proposed to modulate the activity of the enzyme through an allosteric inhibitory mechanism (Tesson et al., 1995). The main aim of this study was to assess the inhibitory effects and nature of the inhibition of imidazol(ine)/guanidine drugs on rat liver MAO-A and MAO-B isoforms and to compare their inhibitory potencies with their affinities for the sites labelled by [3H]-clonidine in the same tissue. 2. Competition for [3H]-clonidine binding in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the pharmacological profile of the interaction (2-styryl-2-imidazoline, LSL 61112 > idazoxan > 2-benzofuranyl-2-imidazoline, 2-BFI = cirazoline > guanabenz > oxymetazoline > > clonidine) was typical of that for I2-sites. 3. Clonidine inhibited rat liver MAO-A and MAO-B activities with very low potency (IC50S: 700 microM and 6 mM, respectively) and displayed the typical pattern of competitive enzyme inhibition (lineweaver-Burk plots: increased K(m) and unchanged Vmax values). Other imidazol(ine)/guanidine drugs also were weak MAO inhibitors with the exception of guanabenz, 2-BFI and cirazoline on MAO-A (IC50S: 4-11 microM) and 2-benzofuranyl-2-imidazol (LSL 60101) on MAO-B (IC50: 16 microM). Idazoxan was a full inhibitor although with rather low potency, on both MAO-A and MAO-B isoenzymes (IC50S: 280 microM and 624 microM, respectively). Kinetic analyses of MAO-A inhibition by these drugs revealed that the interactions were competitive. For the same drugs acting on MAO-B the interactions were of the mixed type inhibition (increased K(m) and decreased Vmax values), although the greater inhibitory effects on the apparent value of Vmax/K(m) than on the Vmax value indicated that the competitive element of the MAO-B inhibition predominated. 4. Competition for [3H]-Ro 41-1049 binding to MAO-A or [3H]-Ro 19-6327 binding to MAO-B in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO-A or MAO-B activities In fact, very good correlations were obtained when the affinities of drugs at MAO-A or MAO-B catalytic sites were correlated with their potencies in inhibiting MAO-A (r = 0.92) or MAO-B (r = 0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO-A and MAO-B isoforms. 5. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low-affinity site (pKiL, micromolar range) of I2-imidazoline receptors labelled with [3H]-clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO-A or MAO-B activity. These discrepancies indicated that I2-imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. 6. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2-imidazoline receptors represent molecular species that are distinct from MAO.

Pharmacological modulation of immunoreactive imidazoline receptor proteins in rat brain: relationship with non-adrenoceptor [3H]-idazoxan binding sites.

1. The densities of various imidazoline receptor proteins (with apparent molecular masses of approximately 29/30-45- and 66-kDa) were quantitated by immunoblotting in the rat cerebral cortex after various drug treatments. The modulation of these imidazoline receptor proteins was then compared with the changes in the density of non-adrenoceptor [3H]-idazoxan binding sites (I2-sites) induced by the same drug treatments. 2. Chronic treatment (7 days) with the I2-selective imidazol(in)e drugs idazoxan (10 mg kg-1), cirazoline (1 mg kg-1) and LSL 60101 (10 mg kg-1) differentially increased the immunoreactivity of imidazoline receptor proteins. The levels of the 29/30-kDa protein were increased by idazoxan and LSL 60101 (23%), the levels of the 45-kDa protein only by cirazoline (44%) and those of the 66-kDa protein only by idazoxan (50%). These drug treatments also increased the density of I2-sites (32-42%). 3. Chronic treatment (7 days) with efaroxan (10 mg kg-1), RX821002 (10 mg kg-1) and yohimbine (10 mg kg-1), which possess very low affinity for I2-imidazoline receptors, did not alter either the immunoreactivity of imidazoline receptor proteins or the density of I2-sites. 4. Chronic treatment (7 days) with the monoamine oxidase (MAO) inhibitors clorgyline (10 mg kg-1) and phenelzine (10 mg kg-1) decreased the immunoreactivity of the 29/30-kDa (17-24%), 45-kDa (19%) and 66-kDa (23-31%) imidazoline receptor proteins. The alkylating agent N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (1.6 mg kg-1, 6 h) also decreased the levels of the three imidazoline receptor proteins (20-47%). These drug treatments consistently decreased the density of I2-sites (31-57%). 5. Significant correlations were found when the mean percentage changes in immunoreactivity of imidazoline receptor proteins were related to the mean percentage changes in the density of I2-sites after the various drug treatments (r = 0.92 for the 29/30-kDa protein, r = 0.69 for the 45-kDa protein and r = 0.75 for the 66-kDa protein). 6. In the rat cerebral cortex the I2-imidazoline receptor labelled by [3H]-idazoxan is heterogeneous in nature and the related imidazoline receptor proteins (29/30-, 45- and 66-kDa) detected by immunoblotting contribute differentially to the modulation of I2-sites after drug treatment.

Activation of I(2)-imidazoline receptors enhances supraspinal morphine analgesia in mice: a model to detect agonist and antagonist activities at these receptors.

This work investigates the receptor acted upon by imidazoline compounds in the modulation of morphine analgesia. The effects of highly selective imidazoline ligands on the supraspinal antinociception induced by morphine in mice were determined. 2. Intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of ligands selective for the I(2)-imidazoline receptor, 2-BFI, LSL 60101, LSL 61122 and aganodine, and the non selective ligand agmatine, increased morphine antinociception in a dose-dependent manner. Neither moxonidine, a mixed I(1)-imidazoline and alpha(2)-adrenoceptor agonist, RX821002, a potent alpha(2)-adrenoceptor antagonist that displays low affinity at I(2)-imidazoline receptors, nor the selective non-imidazoline alpha(2)-adrenoceptor antagonist RS-15385-197, modified the analgesic responses to morphine. 3. Administration of pertussis toxin (0.25 microg per mouse, i.c.v.) 6 days before the analgesic test blocked the ability of the I(2)-imidazoline ligands to potentiate morphine antinociception. 4. The increased effect of morphine induced by I(2)-imidazoline ligands (agonists) was completely reversed by idazoxan and BU 224. Identical results were obtained with IBI, which alkylates I(2)-imidazoline binding sites. Thus, both agonist and antagonist properties of imidazoline ligands at the I(2)-imidazoline receptors were observed. 5. Pre-treatment (30 min) with deprenyl, an irreversible inhibitor of monoamine oxidase B (IMAO-B), produced an increase of morphine antinociception. Clorgyline, an irreversible IMAO-A, given 30 min before morphine did not alter the effect of the opioid. At longer intervals (24 h) a single dose of either clorgyline or deprenyl reduced the density of I(2)-imidazoline receptors and prevented the I(2)-mediated potentiation of morphine analgesia. 6. These results demonstrate functional interaction between I(2)-imidazoline and opioid receptors. The involvement of G(i)-G(o) transducer proteins in this modulatory effect is also suggested.

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L-glutamate (1-100 microM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 microM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 microM (EC100) L-glutamate with the rank order (EC50 in microM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole](101)>RX82 1002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding. In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10-12 microM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.

Pharmacologic and molecular discrimination of I2-imidazoline receptor subtypes.

I2-imidazoline receptors (I2-IR) are characterized by their high affinity for imidazolines and guanidines and medium affinity for imidazolidines. The differential recognition of I2-IR by amiloride led to subtype these sites as amiloride-sensitive (I2A-IR) and amiloride-insensitive (I2B-IR). I2-IR labeled with [3H]idazoxan or [3H]2-BFI in the rabbit cerebral cortex (I2A-IR) displayed higher affinities for amiloride and amiloride analogs than in the rat cerebral cortex (I2B-IR). Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both I2-IR subtypes. The drugs (+)- and (-)-medetomidine, bromoxidine, moxonidine, and clorgyline were more potent on the high and/or low affinity sites of I2B-IR than on I2A-IR. Preincubation (30 min at 25 degrees C) with 10(-6) M isothiocyanatobenzyl imidazoline (IBI) or with 10(-6) M clorgyline reduced by 40% and 26%, respectively, the binding of [3H]2-BFI to I2B-IR, but it did not alter the binding of the radioligand to I2A-IR. These results indicated that the I2-IR subtypes differ in their pharmacologic profiles and in the nature of the imidazoline binding site involved in clorgyline and IBI alkylation. In rat cortical membranes, western blot detection of immunoreactive imidazoline receptor proteins revealed a double band of approximately 29/30 kD and three less intense bands of approximately 45, approximately 66, and approximately 85 kD. In rabbit cortical membranes the antibody detected proteins of approximately 30, approximately 57, approximately 66, and approximately 85 kD. It is suggested that I2-IR may be related to more than one receptor protein and that I2-IR subtypes differ in the nature of the proteins implicated.

Estradiol--induced redistribution of glial fibrillary acidic protein immunoreactivity in the rat brain.

The immunohistochemical distribution of the glial fibrillary acidic protein (GFAP), a marker of glial filaments, was studied on coronal sections of the globus pallidus, the area CA4 of the hippocampus and the arcuate nucleus of the hypothalamus, 3 estrogen-sensitive areas of the rat brain. The number and the surface density of the GFAP-immunoreactive cells were evaluated in 6 adult ovariectomized rats injected with a single dose (20 mg/kg) of estradiol valerate (OVX + E2 rats) and in 6 ovariectomized littermates injected with vehicle (OVX rats). Two days after the injection, a similar distribution of the GFAP was observed in the arcuate nucleus of OVX + E2 rats when compared to OVX rats, whereas a significantly (P less than 0.001) increased surface density of GFAP immunoreactive material was observed in the globus pallidus and hippocampus of estradiol-treated rats. Since the number of GFAP-positive cells was unchanged by the estradiol injection, the enhanced surface density of GFAP immunoreactive material in the hippocampus and globus pallidus suggest a possible influence of estradiol on GFAP-immunoreactive glial processes.