Advancing ocular safety research: A comprehensive examination of benzocaine acute exposure without animal testing.

Benzocaine is a widely employed local anaesthetic; however, there is a notable dearth of preclinical and clinical evidence regarding its safety in ophthalmological products. To address this, a comprehensive strategy incorporating in silico and in vitro methodologies was proposed for assessing benzocaine's ocular toxicity without animal testing. To collect the in silico evidence, the QSAR Toolbox (v4.5) was used. A single exposure to two benzocaine concentrations (2% and 20%) was evaluated by in vitro methods. Hen's Egg Chorioallantoic Membrane Test (HET-CAM) was performed to evaluate the effects on the conjunctiva. To study corneal integrity, Short Time Exposure test (STE) and Bovine Corneal Opacity and Permeability (BCOP) assay, followed by histopathological analysis, were carried out. Results from both in silico and in vitro methodologies categorize benzocaine as non-irritating. The histopathological analysis further affirms the safety of using benzocaine in eye drops, as no alterations were observed in evaluated corneal strata. This research proposes a useful combined strategy to provide evidence on the safety of local anaesthetics and particularly show that 2% and 20% benzocaine solutions do not induce eye irritation or corneal damage, supporting the potential use of benzocaine in the development of ophthalmic anesthetic products.

An enriched environment restores normal behavior while providing cytoskeletal restoration and synaptic changes in the hippocampus of rats exposed to an experimental model of depression.

The exposure of rats to an enriched environment (EE) has several effects in common with the administration of antidepressants. However, there is still little information about the molecular underpinnings of these effects on rats subjected to experimental models of depression. The aim of this research was to evaluate the effects of EE on rats exposed to the learned helplessness paradigm (LH), a well-known model of the disease. We found that a 21 day exposure to EE reverts helplessness behavior to normal in LH animals. Inmunohistochemical labeling showed that this effect was accompanied by normalization of two structural proteins of hippocampal neurons to control values: the light neurofilament subunit (NFL) and the postsynaptic density 95 (PSD-95) protein, which were decreased in LH animals housed in standard cages. The decrease in the presynaptic protein synaptophysin (SYN) observed in LH animals remained unchanged after exposure to EE. There was no increase in neurogenesis as measured by quantification of double-labeled cells with 5-bromo-2'-deoxyuridine (BrdU) and the neuronal marker beta-tubulin class III. These results show that EE may have behavioral and synaptic effects on animals exposed to an experimental model of depression, and that such actions seem to be independent from neurogenesis.

Neuronal cytoskeletal alterations in an experimental model of depression.

It has been proposed that depression is associated with hippocampal morphological changes. The apical dendrite atrophy of hippocampal CA3 pyramidal neurons has been described in experimental models of depression. The aim of the present study was to determine which cytoskeletal components are involved in the morphological changes previously described in the hippocampus of depressed animals. The expression of different neuronal cytoskeletal markers was analyzed by immunohistochemistry in rats exposed to a learned helplessness paradigm, an experimental model of depression. Rats were trained with 60 inescapable foot shocks (0.6 mA/15 s) and escape latencies and failures were tested 4 days after training. Animals in which learned helplessness behavior persisted for 21 days were included in the depressed group. No foot shocks were delivered to control rats. Microtubule-associated protein 2 (MAP-2) and light (NFL; 68 kDa), medium (NFM; 160 kDa) and heavy (NFH; 200 kDa) neurofilament subunit immunostainings were analyzed employing morphometric parameters. In the depressed group, NFL immunostaining decreased 55% (P<0.05) and 60% (P<0.001) in CA3 and dentate gyrus, respectively. In the same areas, MAP-2, NFM and NFH immunostainings did not differ between depressed and control animals. Since NFL is present in the core of mature neurofilament, it is proposed that hippocampal depression-associated plastic alterations may be due to changes in the dynamics of the neurofilament assembly.

Adaptive changes in the rat hippocampal glutamatergic neurotransmission are observed during long-term treatment with lorazepam.

RATIONALE: Chronic treatment with benzodiazepines induces tolerance to most of their pharmacological effects. The best-studied neurochemical correlation to this phenomenon involves GABAergic adaptive changes. However, some compensation by excitatory neurotransmission could also be postulated. OBJECTIVE: The aim of this work was to investigate the effect of chronic treatment with benzodiazepines on several parameters of hippocampal glutamatergic neurotransmission. METHODS: Rats were injected (IP) with a single dose or daily doses (21 days) of 1 mg/kg lorazepam (LZ) or vehicle. Thirty minutes after the last dose, animals were killed and parameters were measured in the dissected hippocampi. We determined one presynaptic parameter, in vitro glutamate release induced by a 60 mM K(+) stimulus. [(3)H]MK-801 binding to postsynaptic NMDA receptors and the NMDA-stimulated efflux of cGMP were also evaluated. RESULTS: While no changes were observed in any of the parameters after a single dose of the drug, we found an increase of 206% in in vitro glutamate release in chronically treated animals [two-way ANOVA: F(1,16)=6.22], together with an increment of 103% in the NMDA-stimulated cGMP efflux [two-way ANOVA: F(1,18)=14.05]. No changes either in K(D) or in B(max) values for [(3)H]MK-801 binding to hippocampal membranes were observed. CONCLUSIONS: Taken together, these changes strongly suggest that a compensatory increase in the glutamatergic response develops in the hippocampus during chronic treatment with LZ. Our findings might indicate a contribution of glutamatergic mechanisms to the tolerance to hippocampal-mediated effects of LZ, such as amnesic and anticonvulsant activities.

Tolerance to the sedative effect of lorazepam correlates with a diminution in cortical release and affinity for glutamate.

Benzodiazepines are anxiolytic, anticonvulsant, sedative and hypnotic compounds usually prescribed on a long-term basis. Chronic treatment with these compounds induces tolerance, which has been extensively attributed to modifications in the GABAergic neurotransmission. However, a compensatory increase in the excitatory response, named as an oppositional response, has also been put forward as a means for explaining such tolerance. Changes in the excitatory neurotransmission have been found in withdrawn rats after a long treatment with benzodiazepines but these modifications have not been conclusively studied during tolerance. In this work we studied several parameters of the glutamatergic neurotransmission in rats made tolerant to the sedative effect of 3 mg/kg (i.p.) of lorazepam (LZ). We found a decrease in the affinity of cortical NMDA receptors for (3)H-glutamate (K(D): 124.4 +/- 13.3 nM in tolerant rats, 71.6 +/- 10.4 nM in controls, P<0.05) together with a decrease in the in vitro 60 mM K(+)-stimulated cortical glutamate release (59+/- 12% vs. 153 +/- 38%, tolerant rats vs. controls, P<0.05). We conclude that tolerance to the sedative effect of LZ correlates with a decreased sensitivity for glutamate that may in turn diminish the cortical response to a chemical stimulus. Our findings constitute an evidence against the oppositional model of pharmacodynamic tolerance in this experimental condition.

Diazepam fails to potentiate GABA-induced chloride uptake and to produce anxiolytic-like action in aged rats.

The pharmacological response to benzodiazepines has been demonstrated to be different in aged individuals in comparison to adults. We studied the age-dependent changes in some of the in vitro and behavioral effects of diazepam in aged (24 months old) rats, comparing them to adults (3 months old). We evaluated the in vitro gamma-aminobutyric acid (GABA)-induced 36Cl- uptake and the diazepam potentiation of GABA-stimulated 36Cl- uptake in microsacs from cerebral cortex of both groups of animals. We found no differences in the GABA-stimulated 36Cl- uptake between adult and aged animals, and diazepam failed to potentiate GABA-induced 36Cl- flux in the aged cortical microsacs. We also examined the effect of 0.03-10 mg of diazepam on locomotor activity in an open-field test and the anxiolytic-like action of diazepam in doses ranging from 0.03 to 1 in a dark-light transition test. We observed no anxiolytic-like action of the drug in the dark-light transition test in the aged rats, while there was a shift to the left in the diminution of locomotor activity evaluated by the open-field test. We conclude that the pharmacodynamic changes observed in cortical GABA(A) receptors in aged rats could partially explain the lack of anxiolytic-like action but not the oversedation evidenced in this group of animals.

[Autobiographic memory. Neurobiological substrates]. / La memoria autobiográfica. Fundamentos neurobiológicos.

This work reviews the cellular mechanisms, and the cerebral areas and circuits participating in the consolidation of episodic memory. At the cerebral level the circuit which connects the primary sensory cortices with the polimodal cortices, the entorhinal cortex, dentate gyrus, hippocampus and associative cortices is sequentially activated when the consolidation of a mnesic trace takes place. Cellular phenomena associated with this process is the long-term potentiation (LTP) which requires the activation of NMDA and AMPA receptors, the uptake of calcium and the activation of several enzymes which on one hand catalize the synthesis of retrograde messengers as nitric oxide and on the other induce genomic changes with trophic consequences for both the post and the presynaptic neurons. Eventual relationships between neurobiological findings and mnesic phenomena observed in psychoanalytic practice should be further investigated.

La memoria autobiográfica. Fundamentos neurobiológicos. / [Autobiographic memory. Neurobiological substrates]

This work reviews the cellular mechanisms, and the cerebral areas and circuits participating in the consolidation of episodic memory. At the cerebral level the circuit which connects the primary sensory cortices with the polimodal cortices, the entorhinal cortex, dentate gyrus, hippocampus and associative cortices is sequentially activated when the consolidation of a mnesic trace takes place. Cellular phenomena associated with this process is the long-term potentiation (LTP) which requires the activation of NMDA and AMPA receptors, the uptake of calcium and the activation of several enzymes which on one hand catalize the synthesis of retrograde messengers as nitric oxide and on the other induce genomic changes with trophic consequences for both the post and the presynaptic neurons. Eventual relationships between neurobiological findings and mnesic phenomena observed in psychoanalytic practice should be further investigated.

La memoria autobiográfica. Fundamentos neurobiológicos / [Autobiographic memory. Neurobiological substrates]

This work reviews the cellular mechanisms, and the cerebral areas and circuits participating in the consolidation of episodic memory. At the cerebral level the circuit which connects the primary sensory cortices with the polimodal cortices, the entorhinal cortex, dentate gyrus, hippocampus and associative cortices is sequentially activated when the consolidation of a mnesic trace takes place. Cellular phenomena associated with this process is the long-term potentiation (LTP) which requires the activation of NMDA and AMPA receptors, the uptake of calcium and the activation of several enzymes which on one hand catalize the synthesis of retrograde messengers as nitric oxide and on the other induce genomic changes with trophic consequences for both the post and the presynaptic neurons. Eventual relationships between neurobiological findings and mnesic phenomena observed in psychoanalytic practice should be further investigated.

Chronic corticosterone impairs inhibitory avoidance in rats: possible link with atrophy of hippocampal CA3 neurons.

The aim of our work was to evaluate the effect of a chronic (22 days) administration of corticosterone, which induces supraphysiological serum levels of the hormone, on an inhibitory avoidance learning in rats (one-trial step-through learning task, footshock: 0.5 mA, 2 s). We also studied hippocampal markers of neuroanatomical CA3 pyramidal neuron atrophy by using the Golgi staining method. Chronic exposure to high CORT serum levels induced a significant impairment of inhibitory avoidance learning. The CORT group also showed hippocampal glucocorticoid receptor (GR) downregulation and the decrease of hippocampal CA3 branch points and total dendritic length in the apical tree that would be causally related with the learning impairment.

Valproic acid differs in its in vitro effect on glutamic acid decarboxylase activity in neonatal and adult rat brain.

1. The in vitro effect of valproic acid (VA) (10(-6) to 10(-3) M) on glutamic acid decarboxylase (GAD) activity in whole brain and cerebral cortex (CC) of neonates and of adult rats was examined. 2. VA did not induce changes on GAD activity either in CC or in the rest of the brain (RB) of adult animals. 3. But at 10(-3) M, VA induced an increase in GAD activity in homogenates of noncortical brain areas of neonates; no increments were found in CC of these animals. This latter increase was detected in the membrane-bound fraction of the enzyme and was not due to physicochemical nonspecific changes related to the potential solvent activity of VA at this high concentration. 4. We may conclude that VA induces changes on GAD activity in neonatal stages of development but not in adult brain. Therefore, although a direct enhancement of GAD activity may play a role in the mechanism of action of VA in pediatric patients, this cannot be verified in the adult population.

Chronic administration of valproic acid induces a decrease in rat striatal glutamate and taurine levels.

The effect of acute and chronic (10 days) administration of 200 mg/kg (i.p.) of valproic acid (VPA) on endogenous levels of aspartate, glutamate, alanine, glycine and taurine in the cerebral frontal cortex and corpus striatum of rats was studied. Quantification of the amino acid levels was performed by HPLC.Valproic acid (VPA) did not either induce changes on these neurotransmitters contents in corpus striatum after acute treatment. After chronic administration we found a decrease on the endogenous levels of glutamic acid (24%, p < 0.05) which was related to an increase (250%, p < 0.02) of the in vitro KCl evoked release of glutamate. We found decrements in taurine endogenous levels (22%, p < 0.05) which was not associated with an increase of its release.In cerebral frontal cortex there was not found any change neither under the acute nor under the chronic condition.Thus, it may be conclude that chronic treatment with VPA produces decreases on the endogenous levels of glutamate and taurine. However the relevance of this effect concerning it therapeutic action remains unclear.

The glucocorticoid sensitivity of lymphocytes changes according to the activity of the hypothalamic-pituitary-adrenocortical system.

Cortisol inhibition of phytohemagglutinin (PHA)-induced lymphocyte activation was studied in vitro in peripheral blood mononuclear cells (PBMC) of patients with pathologies of the hypothalamic-pituitary-adrenocortical (HPA) system (n = 18) as well as in students under and out of academic stress. In patients with HPA pathologies, cortisol-induced inhibition of lymphocyte interleukin-2 (IL-2) secretion in vitro positively correlated (p < .005) with IL-2 synthesis, such that lymphocytes secreting less IL-2 were less cortisol sensitive. Similarly, in stressed students there was a positive correlation (p < .02) between lymphocyte DNA synthesis and cortisol inhibition in vitro. Academic stress also resulted in a decrease (p < .01) of lymphocyte cortisol sensitivity. The same tendency was observed in patients with Cushing's syndrome when compared with hypocortisolemic patients. Normal controls whose lymphocytes were glucocorticoid resistant showed higher lymphocyte activation than glucocorticoid sensitive subjects. This pattern of glucocorticoid sensitivity was reversed in individuals with abnormal glucocorticoid secretion due to HPA pathology or stress.

[Role of excitatory amino acids in neuropathology]. / Papel de los aminoácidos excitatorios en la neuropatología.

Excitatory amino acids (EAA) became known as neurotransmitters of the central nervous system (CNS) in the last decade. The most studied EAA are glutamate and aspartate. Both are synthetized by the same mechanism as gamaaminobutyric acid. (Fig. 1). Glutamate is widely distributed in the CNS and the spinal cord, being the areas of higher concentration the cerebral cortex, the hypocampus and the cerebellum. There have been identified two type of receptors for glutamate: ionotropic and metabotropic. The former includes three different types: NMDA, AMPA and KA. NMDA receptor is coupled to a Na+ and Ca2+ channel being the second ion the most important one. This receptor has several sites of binding for various substances. Along with the site for N-methyl-D-aspartate, which binds glutamate and/or aspartate, there have been identified a site for the binding of glycine (which is different from the strychnine sensitive one), a site for poliamines such as spermine and spermidine, and a site for the binding of Zn2+ (Table 1). AMPA receptor is associated to a Ca(2+)-Na+ channel, being in this case the Na+ the most important ion. There are two metabotropic type receptors: L-AP4 and trans-ACPD. Both are coupled to a G protein and agonists exert their action increasing phospholipase C activity which in turn induces an increment of IP3 and diacyl-glicerol, and a consecutive releasing of Ca2+ from intracellular stores. EAA play a role in some physiological processes. One of them is long-term potentiation (LTP), an electrochemical phenomenon involved in memory consolidation. Antagonists of NMDA and AMPA receptor prevent the development of LTP, and conversely, the agonist of glycine site of NMDA receptor--D-cycloserine--facilitates memory consolidation. Since 1957, EAA are considered neurotoxic substances and there are many indirect evidences to support this statement. Pathogenesis of neuronal damage elicited by EAA involves the events shown in Fig. 3. Prevention of the cascade of events that provokes neurotoxicity may be achieved by NMDA antagonists, but once it has begun it may be only aborted subtracting the Ca2+ from the medium, using nifedipine or blocking AMPA receptor with an antagonist (CNQX). EAA have been shown to play a toxic role in neuronal damage induced by ischemia. Research using various experimental models demonstrated that NMDA receptor antagonists (i.e. MK 801) blocks postischemic damage. Interventions at various levels of the pathogenic cascade shown in Fig. 4 provoke the same results. There is enough evidence to suspect that NMDA and AMPA receptors are altered in epilepsy. NMDA antagonists (i.e. MK801 or AP5) prevent the development of epileptic seizures induced by kindling; CNQX, an AMPA antagonist, blocks the increase in electrical activity induced by K+ in slices of hypocampus; felbamate, an antiepileptic drug, blocks the glycine site (not strychnine sensitive) decreasing NMDA receptor activity. Several neurodegenerative disorders have been associated with exogenous administration or accidental intake of EAA. (i.e. neurolatirism, Guam disease). Similarities between these diseases and lateral aminotrophic sclerosis indicate that in the latter EAA may play a pathogenic role. Finally, the psychotomimetic effect of phencyclidine (an antagonist of NMDA receptor) suggests that in schizophrenia, together with dopaminergic neurotransmission impairment, some dysfunction of glutamate pathways may be present.

Papel de los aminoácidos excitatorios en la neuropatología. / [Role of excitatory amino acids in neuropathology]

Excitatory amino acids (EAA) became known as neurotransmitters of the central nervous system (CNS) in the last decade. The most studied EAA are glutamate and aspartate. Both are synthetized by the same mechanism as gamaaminobutyric acid. (Fig. 1). Glutamate is widely distributed in the CNS and the spinal cord, being the areas of higher concentration the cerebral cortex, the hypocampus and the cerebellum. There have been identified two type of receptors for glutamate: ionotropic and metabotropic. The former includes three different types: NMDA, AMPA and KA. NMDA receptor is coupled to a Na+ and Ca2+ channel being the second ion the most important one. This receptor has several sites of binding for various substances. Along with the site for N-methyl-D-aspartate, which binds glutamate and/or aspartate, there have been identified a site for the binding of glycine (which is different from the strychnine sensitive one), a site for poliamines such as spermine and spermidine, and a site for the binding of Zn2+ (Table 1). AMPA receptor is associated to a Ca(2+)-Na+ channel, being in this case the Na+ the most important ion. There are two metabotropic type receptors: L-AP4 and trans-ACPD. Both are coupled to a G protein and agonists exert their action increasing phospholipase C activity which in turn induces an increment of IP3 and diacyl-glicerol, and a consecutive releasing of Ca2+ from intracellular stores. EAA play a role in some physiological processes. One of them is long-term potentiation (LTP), an electrochemical phenomenon involved in memory consolidation. Antagonists of NMDA and AMPA receptor prevent the development of LTP, and conversely, the agonist of glycine site of NMDA receptor--D-cycloserine--facilitates memory consolidation. Since 1957, EAA are considered neurotoxic substances and there are many indirect evidences to support this statement. Pathogenesis of neuronal damage elicited by EAA involves the events shown in Fig. 3. Prevention of the cascade of events that provokes neurotoxicity may be achieved by NMDA antagonists, but once it has begun it may be only aborted subtracting the Ca2+ from the medium, using nifedipine or blocking AMPA receptor with an antagonist (CNQX). EAA have been shown to play a toxic role in neuronal damage induced by ischemia. Research using various experimental models demonstrated that NMDA receptor antagonists (i.e. MK 801) blocks postischemic damage. Interventions at various levels of the pathogenic cascade shown in Fig. 4 provoke the same results. There is enough evidence to suspect that NMDA and AMPA receptors are altered in epilepsy. NMDA antagonists (i.e. MK801 or AP5) prevent the development of epileptic seizures induced by kindling; CNQX, an AMPA antagonist, blocks the increase in electrical activity induced by K+ in slices of hypocampus; felbamate, an antiepileptic drug, blocks the glycine site (not strychnine sensitive) decreasing NMDA receptor activity. Several neurodegenerative disorders have been associated with exogenous administration or accidental intake of EAA. (i.e. neurolatirism, Guam disease). Similarities between these diseases and lateral aminotrophic sclerosis indicate that in the latter EAA may play a pathogenic role. Finally, the psychotomimetic effect of phencyclidine (an antagonist of NMDA receptor) suggests that in schizophrenia, together with dopaminergic neurotransmission impairment, some dysfunction of glutamate pathways may be present.

Lack of anxiolytic effect of diazepam in pre-anaesthetic medication.

In 30 female patients undergoing elective cholecystectomy, we have compared the anxiolytic effect of diazepam 10 mg with placebo, using measurements of cardiovascular (arterial pressure and heart rate), biochemical (plasma concentrations of noradrenaline, adrenaline, cortisol and dopamine beta-hydroxylase), subjective (visual analogue scale) and behavioural (Hamilton anxiety test) variables. Pretreatment evaluation was carried out the day before surgery and post-treatment examination was performed 1.5-2 h after oral administration of premedication, immediately before transfer of patients to the operating room. We found that diazepam 10 mg had no significant effect on any of the measured variables.