Determinants of clinical response to empirical antibiotic treatment in patients with cirrhosis and bacterial and fungal infections-Results from the ICA "Global Study" (EABCIR-Global Study).

BACKGROUND: The administration of an appropriate empirical antibiotic treatment is essential in cirrhosis and severe bacterial infections. We aimed to investigate the predictors of clinical response of empirical antibiotic treatment in a prospective cohort of patients with cirrhosis and bacterial and fungal infections included in the International Club of Ascites "Global Study." METHODS: Patients hospitalized with cirrhosis and bacterial/fungal infection were prospectively enrolled at 46 centers. Clinical response to antibiotic treatment was defined according to changes in markers of infection/inflammation, vital signs, improvement of organ failure, and results of cultures. RESULTS: From October 2015 to September 2016, 1302 patients were included at 46 centers. A clinical response was achieved in only 61% of cases. Independent predictors of lack of clinical response to empirical treatment were C-reactive protein (OR = 1.16; 95% CI = 1.02-1.31), blood leukocyte count (OR = 1.39;95% CI = 1.09-1.77), serum albumin (OR = 0.70; 95% CI = 0.55-0.88), nosocomial infections (OR = 1.96; 95% CI = 1.20-2.38), pneumonia (OR = 1.75; 95% CI = 1.22-2.53), and ineffective treatment according to antibiotic susceptibility test (OR = 5.32; 95% CI = 3.47-8.57). Patients with a lack of clinical response to first-line antibiotic treatment had a significantly lower resolution rate of infections (55% vs. 96%; p < 0.001), a higher incidence of second infections (29% vs. 15%; p < 0.001), shock (35% vs. 7%; p < 0.001) and new organ failures (52% vs. 19 %; p < 0.001) than responders. Clinical response to empirical treatment was an independent predictor of 28-day survival ( subdistribution = 0.20; 95% CI = 0.14-0.27). CONCLUSIONS: Four out of 10 patients with cirrhosis do not respond to the first-line antibiotic therapy, leading to lower resolution of infections and higher mortality. Broader-spectrum antibiotics and strategies targeting systemic inflammation may improve prognosis in patients with a high degree of inflammation, low serum albumin levels, and severe liver impairment.

Genetic Ancestry, Race, and Severity of Acutely Decompensated Cirrhosis in Latin America.

BACKGROUND & AIMS: Genetic ancestry or racial differences in health outcomes exist in diseases associated with systemic inflammation (eg, COVID-19). This study aimed to investigate the association of genetic ancestry and race with acute-on-chronic liver failure (ACLF), which is characterized by acute systemic inflammation, multi-organ failure, and high risk of short-term death. METHODS: This prospective cohort study analyzed a comprehensive set of data, including genetic ancestry and race among several others, in 1274 patients with acutely decompensated cirrhosis who were nonelectively admitted to 44 hospitals from 7 Latin American countries. RESULTS: Three hundred ninety-five patients (31.0%) had ACLF of any grade at enrollment. Patients with ACLF had a higher median percentage of Native American genetic ancestry and lower median percentage of European ancestry than patients without ACLF (22.6% vs 12.9% and 53.4% vs 59.6%, respectively). The median percentage of African genetic ancestry was low among patients with ACLF and among those without ACLF. In terms of race, a higher percentage of patients with ACLF than patients without ACLF were Native American and a lower percentage of patients with ACLF than patients without ACLF were European American or African American. In multivariable analyses that adjusted for differences in sociodemographic and clinical characteristics, the odds ratio for ACLF at enrollment was 1.08 (95% CI, 1.03-1.13) with Native American genetic ancestry and 2.57 (95% CI, 1.84-3.58) for Native American race vs European American race CONCLUSIONS: In a large cohort of Latin American patients with acutely decompensated cirrhosis, increasing percentages of Native American ancestry and Native American race were factors independently associated with ACLF at enrollment.

Development and external validation of a model to predict multidrug-resistant bacterial infections in patients with cirrhosis.

With the increasing rate of infections caused by multidrug-resistant organisms (MDRO), selecting appropriate empiric antibiotics has become challenging. We aimed to develop and externally validate a model for predicting the risk of MDRO infections in patients with cirrhosis. METHODS: We included patients with cirrhosis and bacterial infections from two prospective studies: a transcontinental study was used for model development and internal validation (n = 1302), and a study from Argentina and Uruguay was used for external validation (n = 472). All predictors were measured at the time of infection. Both culture-positive and culture-negative infections were included. The model was developed using logistic regression with backward stepwise predictor selection. We externally validated the optimism-adjusted model using calibration and discrimination statistics and evaluated its clinical utility. RESULTS: The prevalence of MDRO infections was 19% and 22% in the development and external validation datasets, respectively. The model's predictors were sex, prior antibiotic use, type and site of infection, MELD-Na, use of vasopressors, acute-on-chronic liver failure, and interaction terms. Upon external validation, the calibration slope was 77 (95% CI .48-1.05), and the area under the ROC curve was .68 (95% CI .61-.73). The application of the model significantly changed the post-test probability of having an MDRO infection, identifying patients with nosocomial infection at very low risk (8%) and patients with community-acquired infections at significant risk (36%). CONCLUSION: This model achieved adequate performance and could be used to improve the selection of empiric antibiotics, aligning with other antibiotic stewardship program strategies.

Clinical features and evolution of bacterial infection-related acute-on-chronic liver failure.

BACKGROUND & AIMS: Bacterial infections can trigger the development of organ failure(s) and acute-on-chronic liver failure (ACLF). Geographic variations in bacteriology and clinical practice could lead to worldwide differences in ACLF epidemiology, phenotypes and associated outcomes. Herein, we aimed to evaluate regional differences in bacterial infection-related ACLF in patients with cirrhosis admitted to hospital. METHODS: This post hoc analysis included 1,175 patients with decompensated cirrhosis (with bacterial infection on admission or nosocomial infection) from 6 geographic regions worldwide. Clinical, laboratory and microbiological data were collected from the diagnosis of infection. Patients were followed-up for organ failure(s) and ACLF development according to the EASL-CLIF criteria from enrolment to discharge/death. RESULTS: A total of 333 patients (28%) had ACLF at diagnosis of infection, while 230 patients developed ACLF after diagnosis of infection, resulting in an overall rate of bacterial infection related-ACLF of 48%, with rates differing amongst different geographic regions (38% in Southern Europe vs. 75% in the Indian subcontinent). Bacterial infection related-ACLF more frequently developed in younger patients (55 ± 13 vs. 58 ± 14 years), males (73% vs. 62%), patients with alcohol-related cirrhosis (59% vs. 45%) and those with a higher baseline MELD score (25 ± 11 vs. 16 ± 5) (all p <0.001). Spontaneous bacterial peritonitis, pneumonia or infections caused by extensively drug resistant (XDR) bacteria were more frequently associated with ACLF development. More patients with ACLF had a positive quick sequential organ failure assessment score and septic shock, resulting in a lower infection resolution rate (all p <0.001). CONCLUSIONS: Bacterial infections, especially with XDR organisms, are associated with the highest risk of ACLF development, accounting for almost half of cases globally. Geographic differences result in variable epidemiology and clinical outcomes. LAY SUMMARY: Bacterial infections can trigger a sudden deterioration in an otherwise stable cirrhotic patient, a condition known as acute-on-chronic liver failure or ACLF. This study has found that the development of ACLF following bacterial infection occurs most commonly in the Indian subcontinent and less so in Southern Europe. The common infections that can trigger ACLF include infection of the abdominal fluid, known as spontaneous bacterial peritonitis, pneumonia and by bacteria that are resistant to multiple antibiotics. Patients who develop ACLF following a bacterial infection have high death rates and are frequently unable to clear the infection.

Prospective Latin American cohort evaluating outcomes of patients with COVID-19 and abnormal liver tests on admission.

INTRODUCTION & OBJECTIVES: The independent effect of liver biochemistries as a prognostic factor in patients with COVID-19 has not been completely addressed. We aimed to evaluate the prognostic value of abnormal liver tests on admission of hospitalized patients with COVID-19. MATERIALS & METHODS: We performed a prospective cohort study including 1611 hospitalized patients with confirmed SARS-CoV-2 infection from April 15, 2020 through July 31, 2020 in 38 different Hospitals from 11 Latin American countries. We registered clinical and laboratory parameters, including liver function tests, on admission and during hospitalization. All patients were followed until discharge or death. We fit multivariable logistic regression models, further post-estimation effect through margins and inverse probability weighting. RESULTS: Overall, 57.8% of the patients were male with a mean age of 52.3 years, 8.5% had chronic liver disease and 3.4% had cirrhosis. Abnormal liver tests on admission were present on 45.2% (CI 42.7-47.7) of the cohort (n = 726). Overall, 15.1% (CI 13.4-16.9) of patients died (n = 244). Patients with abnormal liver tests on admission presented higher mortality 18.7% (CI 15.9-21.7), compared to those with normal liver biochemistries 12.2% (CI 10.1-14.6); P < .0001). After excluding patients with history of chronic liver disease, abnormal liver tests on admission were independently associated with death [OR 1.5 (CI 1.1-2.0); P = 0.01], and severe COVID-19 (2.6 [2.0-3.3], P < .0001), both adjusted by age, gender, diabetes, pneumonia and body mass index >30. CONCLUSIONS: The presence of abnormal liver tests on admission is independently associated with mortality and severe COVID-19 in hospitalized patients with COVID-19 infection and may be used as surrogate marker of inflammation. CLINICALTRIALS.GOV: NCT04358380.

Epidemiology and Effects of Bacterial Infections in Patients With Cirrhosis Worldwide.

BACKGROUND & AIMS: Bacterial infections are common and life-threatening in patients with cirrhosis. Little is known about the epidemiology of bacterial infections in different regions. We performed a multicenter prospective intercontinental study to assess the prevalence and outcomes of bacterial and fungal infections in patients with cirrhosis. METHODS: We collected data from 1302 hospitalized patients with cirrhosis and bacterial or fungal infections at 46 centers (15 in Asia, 15 in Europe, 11 in South America, and 5 in North America) from October 2015 through September 2016. We obtained demographic, clinical, microbiology, and treatment data at time of diagnosis of infection and during hospitalization. Patients were followed until death, liver transplantation, or discharge. RESULTS: The global prevalence of multidrug-resistant (MDR) bacteria was 34% (95% confidence interval 31%-37%). The prevalence of MDR bacteria differed significantly among geographic areas, with the greatest prevalence in Asia. Independent risk factors for infection with MDR bacteria were infection in Asia (particularly in India), use of antibiotics in the 3 months before hospitalization, prior health care exposure, and site of infection. Infections caused by MDR bacteria were associated with a lower rate of resolution of infection, a higher incidence of shock and new organ failures, and higher in-hospital mortality than those caused by non-MDR bacteria. Administration of adequate empirical antibiotic treatment was independently associated with improved in-hospital and 28-day survival. CONCLUSIONS: In a worldwide study of hospitalized patients, we found a high prevalence of infection with MDR bacteria in patients with cirrhosis. Differences in the prevalence of MDR bacterial infections in different global regions indicate the need for different empirical antibiotic strategies in different continents and countries. While we await new antibiotics, effort should be made to decrease the spread of MDR bacteria in patients with cirrhosis.

Neurochemistry and circuit organization of the lateral spiriform nucleus of birds: A uniquely nonmammalian direct pathway component of the basal ganglia.

We used diverse methods to characterize the role of avian lateral spiriform nucleus (SpL) in basal ganglia motor function. Connectivity analysis showed that SpL receives input from globus pallidus (GP), and the intrapeduncular nucleus (INP) located ventromedial to GP, whose neurons express numerous striatal markers. SpL-projecting GP neurons were large and aspiny, while SpL-projecting INP neurons were medium sized and spiny. Connectivity analysis further showed that SpL receives inputs from subthalamic nucleus (STN) and substantia nigra pars reticulata (SNr), and that the SNr also receives inputs from GP, INP, and STN. Neurochemical analysis showed that SpL neurons express ENK, GAD, and a variety of pallidal neuron markers, and receive GABAergic terminals, some of which also contain DARPP32, consistent with GP pallidal and INP striatal inputs. Connectivity and neurochemical analysis showed that the SpL input to tectum prominently ends on GABAA receptor-enriched tectobulbar neurons. Behavioral studies showed that lesions of SpL impair visuomotor behaviors involving tracking and pecking moving targets. Our results suggest that SpL modulates brainstem-projecting tectobulbar neurons in a manner comparable to the demonstrated influence of GP internus on motor thalamus and of SNr on tectobulbar neurons in mammals. Given published data in amphibians and reptiles, it seems likely the SpL circuit represents a major direct pathway-type circuit by which the basal ganglia exerts its motor influence in nonmammalian tetrapods. The present studies also show that avian striatum is divided into three spatially segregated territories with differing connectivity, a medial striato-nigral territory, a dorsolateral striato-GP territory, and the ventrolateral INP motor territory.

Acute cocaine treatment increases thimet oligopeptidase in the striatum of rat brain.

Many studies indicate that thimet oligopeptidase (EC3.4.24.15; TOP) can be implicated in the metabolism of bioactive peptides, including dynorphin 1-8, α-neoendorphin, ß-neoendorphin and GnRH. Furthermore, the higher levels of this peptidase are found in neuroendocrine tissue and testis. In the present study, we have evaluated the effect of acute cocaine administration in male rats on TOP specific activity and mRNA levels in prosencephalic brain areas related with the reward circuitry; ventral striatum, hippocampus, and frontal cortex. No significant differences on TOP specific activity were detected in the hippocampus and frontal cortex of cocaine treated animals compared to control vehicle group. However, a significant increase in activity was observed in the ventral striatum of cocaine treated-rats. The increase occurred in both, TOP specific activity and TOP relative mRNA amount determined by real time RT-PCR. As TOP can be implicated in the processing of many neuropeptides, and previous studies have shown that cocaine also alters the gene expression of proenkephalin and prodynorphin in the striatum, the present findings suggest that TOP changes in the brain could play important role in the balance of neuropeptide level correlated with cocaine effects.

GaAs 904-nm laser irradiation improves myofiber mass recovery during regeneration of skeletal muscle previously damaged by crotoxin.

This work investigated the effect of gallium arsenide (GaAs) irradiation (power: 5 mW; intensity: 77.14 mW/cm(2), spot: 0.07 cm(2)) on regenerating skeletal muscles damaged by crotoxin (CTX). Male C57Bl6 mice were divided into six groups (n = 5 each): control, treated only with laser at doses of 1.5 J or 3 J, CTX-injured and, CTX-injured and treated with laser at doses of 1.5 J or 3 J. The injured groups received a CTX injection into the tibialis anterior (TA) muscle. After 3 days, TA muscles were submitted to GaAs irradiation at doses of 1.5 or 3 J (once a day, during 5 days) and were killed on the eighth day. Muscle histological sections were stained with hematoxylin and eosin (H&E) in order to determine the myofiber cross-sectional area (CSA), the previously injured muscle area (PIMA) and the area density of connective tissue. The gene expression of MyoD and myogenin was detected by real-time PCR. GaAs laser at a dose of 3 J, but not 1.5 J, significantly increased the CSA of regenerating myofibers and reduced the PIMA and the area density of intramuscular connective tissue of CTX-injured muscles. MyoD gene expression increased in the injured group treated with GaAs laser at a dose of 1.5 J. The CTX-injured, 3-J GaAs laser-treated, and the CTX-injured and treated with 3-J laser groups showed an increase in myogenin gene expression when compared to the control group. Our results suggest that GaAs laser treatment at a dose of 3 J improves skeletal muscle regeneration by accelerating the recovery of myofiber mass.

Pigeon as a model to study peripheral projections from the horizontal semicircular canal vestibular apparatus to a brainstem target immunoreactive for AMPA.

PURPOSE: To evaluate whether the pigeon (Columba livia) is a good model for evaluating the vestibular system involved with postural maintenance during movement. METHODS: This study maps the brainstem targets of the horizontal ampullary inputs from the vestibular periphery of the pigeon. We used biotin dextran amine (BDA) injection in horizontal semicircular canal (HSCC), immunohistochemistry for GluR2/3 and GluR4 AMPA and computerized histomorphology reconstruction. RESULTS: Our results show the same distribution pattern with ipsilateral projections to vestibular nuclear complex (VNC) from the HSCC, with the majority of labeled fibers being, long, thin, with few varicosities and many ramifications. Horizontal semicircular canal projections achieve neurons belonging to all nuclei of the VNC with exception of dorsal portion of lateral vestibular nucleus and this area express GluR2/3 and GluR4 AMPA receptors reinforcing the idea of glutamate participation in these connections. CONCLUSIONS: Pigeon is an appropriated experimental model to study of projections of HSCC and reinforcing the information that the vestibular system has strong relation with the fast responses necessary for postural control. Moreover, its phylogenetic organization apparently conservation, also seems to be a fundamental characteristic for vertebrates.

Characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of Helicobacter pylori lipopolysaccharide.

BACKGROUND: The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6-glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6-glucan and characterize their binding properties and functional activity. MATERIALS AND METHODS: BALB/c mice were injected intraperitoneally with 10(8) formalin-fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti-α1,6-glucan-producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole-cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild-type and mutant strains of H. pylori. RESULTS: The generated anti-α1,6-glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6-linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD450 ≥ 0.2). Bactericidal activity was observed against selective wild-type and mutant H. pylori strains exhibiting OD450 values of ≥ 0.45 in WCE. CONCLUSIONS: Anti-α1,6-glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS-based vaccine against H. pylori infections.

Isolation of Helicobacter pylori in gastric mucosa and susceptibility to five antimicrobial drugs in Southern chile.

Helicobacter pylori colonizes more than 50% of the world population thus, it is considered an important cause of gastric cancer. The aim of this study was to determine the isolation frequency of H. pylori in Southern Chile from patients with symptomatology compatible with gastritis or gastric ulcer and to correlate these findings with demographic parameters of infected patients and the susceptibility profiles of the isolated strains to the antimicrobial drugs used in the eradication treatments. A total of 240 patients were enrolled in the study. Each gastric biopsy was homogenized and seeded onto blood agar plates containing a selective antibiotics mixture (DENT supplement). Plates were incubated at 37° C in a microaerophilic environment for five days. The susceptibility profiles to amoxicillin, ciprofloxacin, clarithromycin, tetracycline and metronidazole were determined using the E-test method. H. pylori was isolated from 99 patients (41.3%) with slightly higher frequency in female (42% positive cultures) than male (40.2% positive cultures). With regard to age and educational level, the highest isolation frequencies were obtained in patients between 21-30 (55%) and 41-50 (52.6%) years old, and patients with secondary (43.9%) and university (46.2%) educational levels. Nineteen (21.6%) strains showed resistance to at least one antimicrobial drug. Tetracycline was the most active antimicrobial in vitro, whereas metronidazole was the less active. One strain (5.3%) showed resistance to amoxicillin, clarithomycin and metronidazole, simultaneously.

Convolutional Neural Networks Using Enhanced Radiographs for Real-Time Detection of Sitophilus zeamais in Maize Grain.

The application of artificial intelligence (AI) such as deep learning in the quality control of grains has the potential to assist analysts in decision making and improving procedures. Advanced technologies based on X-ray imaging provide markedly easier ways to control insect infestation of stored products, regardless of whether the quality features are visible on the surface of the grains. Here, we applied contrast enhancement algorithms based on peripheral equalization and calcification emphasis on X-ray images to improve the detection of Sitophilus zeamais in maize grains. In addition, we proposed an approach based on convolutional neural networks (CNNs) to identity non-infested and infested classes using three different architectures; (i) Inception-ResNet-v2, (ii) Xception and (iii) MobileNetV2. In general, the prediction models developed based on the MobileNetV2 and Xception architectures achieved higher accuracy (≥0.88) in identifying non-infested grains and grains infested by maize weevil, with a correct classification from 0.78 to 1.00 for validation and test sets. Hence, the proposed approach using enhanced radiographs has the potential to provide precise control of Sitophilus zeamais for safe human consumption of maize grains. The proposed method can automatically recognize food contaminated with hidden storage pests without manual features, which makes it more reliable for grain inspection.

Morphometric analysis of the AMPA-type neurons in the Deiter's vestibular complex of the chick brain.

Chicken (Gallus gallus) brains were used to investigate the typology and the immunolabel pattern for the subunits composing the AMPA-type glutamate receptors (GluR) of hindbrain neurons of the dorsal (dND) and ventral nuclei (vND) of the Deiter's vestibular complex (CD), which is the avian correspondent of the lateral vestibular nucleus (LVN) of mammals. Our results revealed that neurons of both divisions were poor in GluR1. The vND, the GluR2/3+ and GluR4+ label presented no area or neuronal size preference, although most neurons were around 75%. The dND neurons expressing GluR2/3 are primarily around 85%, medium to large-sized 85%, and predominantly 60% located in the medial portion of the rostral pole and in the lateral portion of the caudal pole. The majority of dND neurons containing GluR4 are also around 75%, larger (70% are large and giant), exhibiting a distribution that seems to be complementary to that of GluR2/3+ neurons. This distinct arrangement indicates functional differences into and between the DC nuclei, also signaling that such variation could be attributed to the diverse nature of the subunit composition of the GluRs. Discussion addresses the morphological and functional correlation of the avian DC with the LVN of mammals in addition to the high morphological correspondence, To include these data into the modern comparative approach we propose to adopt a similar nomenclature for the avian divisions dND and vND that could be referred as dLVN and vLVN.

Analysis of Helicobacter pylori isolates from Chile: occurrence of selective type 1 Lewis b antigen expression in lipopolysaccharide.

Previous studies have shown that the LPS of Helicobacter pylori isolated from North American and European hosts predominantly expresses type 2 Lewis x (Le(x)) and Le(y) epitopes, whilst the LPS from Asian strains has the capacity to express type 1 Le(a) and Le(b) structures. The aim of this study was to evaluate the expression of Le antigens and the cytotoxin-associated antigen (CagA) by H. pylori isolates from Chile. A total of 38 isolates were screened. The expression of Le antigens and CagA was determined by whole-cell indirect ELISA, using commercially available monoclonal anti-Le and polyclonal anti-CagA antibodies. LPS profiles of H. pylori isolates were assessed by gel electrophoresis and Western blotting. Expression of Le(x) and/or Le(y) epitopes was confirmed in 32/38 isolates (84 %), whilst 9/38 isolates (24 %) expressed type 1 Le(b) blood group determinants, in addition to type 2 Le(x) and Le(y) structures. Six strains (16 %) were non-typeable. The majority of H. pylori strains examined were CagA-positive (83.3 %).

Discrete retinal input to the parabrachial complex of a new-world primate, the common marmoset (Callithrix jacchus).

Traditional retinal projections target three functionally complementary systems in the brain of mammals: the primary visual system, the visuomotor integration systems and the circadian timing system. In recent years, studies in several animals have been conducted to investigate the retinal projections to these three systems, despite some evidence of additional targets. The aim of this study was to disclose a previously unknown connection between the retina and the parabrachial complex of the common marmoset, by means of the intraocular injection of cholera toxin subunit b. A few labeled retinal fibers/terminals that are detected in the medial parabrachial portion of the marmoset brain show clear varicosities, suggesting terminal fields. Although the possible role of these projections remains unknown, they may provide a modulation of the cholinergic parabrachial neurons which project to the thalamic dorsal lateral geniculate nucleus.

Involvement of urocortinergic neurons below the midbrain central gray in the physiological response to restraint stress in pigeons.

The present study was carried out to identify the diencephalic and midbrain neurons in pigeons that respond to stress (using restraint as the stressor) and determine if the urocortinergic neurons (expressing urocortin 1, Ucn1) below the midbrain central gray are among those activated. Immunolabeling for the immediate early gene Egr-1 was used to identity stress-responsive neurons, following 1-3 h of restraint. A large increase in nuclear Egr-1 immunolabeling was observed in several dorsomedial thalamic nuclei, and in a stream of neurons extending from below the mesencephalic central gray (overlapping the nucleus of Darkschewitsch at these levels) to just anterior to the nucleus of Edinger-Westphal. A more modest increase in neuronal nuclear Egr-1 was observed in the medial posterior hypothalamic area, the mesencephalic periventricular area, the ventral tegmental area, the inferior colliculus, the nucleus paramedianus of the midbrain, and the intercollicular nucleus. The distribution and abundance of urocortin-immunolabeled neurons coincided with that of the stress-responsive neurons below the mesencephalic periaqueductal gray, and about 50% of these urocortin neurons were activated by stress. These results suggest that, as in some mammals, the urocortinergic neurons of the paramedian subgriseal mesencephalon respond to stress. In those mammals, in which the boundaries of the nucleus of Edinger-Westphal are indistinct, the caudal part of the homologous field of urocortinergic neurons has been referred to as the nucleus of Edinger-Westphal. In pigeons, in which the nucleus of Edinger-Westphal is cytoarchitectonically well-defined, the caudal part of this urocortinergic field clearly does not include the nucleus of Edinger-Westphal.

Pigeon as a model to study peripheral projections from the horizontal semicircular canal vestibular apparatus to a brainstem target immunoreactive for AMPA

ABSTRACT Purpose: To evaluate whether the pigeon (Columba livia) is a good model for evaluating the vestibular system involved with postural maintenance during movement. Methods: This study maps the brainstem targets of the horizontal ampullary inputs from the vestibular periphery of the pigeon. We used biotin dextran amine (BDA) injection in horizontal semicircular canal (HSCC), immunohistochemistry for GluR2/3 and GluR4 AMPA and computerized histomorphology reconstruction. Results: Our results show the same distribution pattern with ipsilateral projections to vestibular nuclear complex (VNC) from the HSCC, with the majority of labeled fibers being, long, thin, with few varicosities and many ramifications. Horizontal semicircular canal projections achieve neurons belonging to all nuclei of the VNC with exception of dorsal portion of lateral vestibular nucleus and this area express GluR2/3 and GluR4 AMPA receptors reinforcing the idea of glutamate participation in these connections. Conclusions: Pigeon is an appropriated experimental model to study of projections of HSCC and reinforcing the information that the vestibular system has strong relation with the fast responses necessary for postural control. Moreover, its phylogenetic organization apparently conservation, also seems to be a fundamental characteristic for vertebrates.

Differential effects of aging on the distribution of calcium-binding proteins in a pretectal nucleus of the chicken brain.

The nucleus pretectalis (PT) of birds is an ovoid-shaped visuomotor cell group of the pretectum that receives tectal input and projects back to the optic tectum. We performed immunohistochemical single- and double-labeling to determine the distribution and abundance of neurons containing three calcium-binding proteins, parvalbumin (PV), calretinin (CR), and calbindin (CB), in the PT in chickens at three ages. We found that PV-positive and CR-positive cells co-localize and are largely found in the outer part of PT at all ages. The GluR4 subunit of the AMPA-type glutamate receptor was selectively localized to these neurons. CB-positive neurons, however, were largely absent from the PT in young and adult chickens. The abundance of PV-positive and CR-positive neurons in PT in old birds was indistinguishable from that in the younger birds, but CB-positive perikarya were 10-20-fold more common than in young birds, and were again mainly found in the outer part of PT. The overall abundance of neurons in PT was reduced to about 50% of its former abundance in the old birds, with this loss restricted to the central part of the nucleus. These data indicate that a cell loss process develops in PT as birds age, that parvalbuminergic and calretinergic neurons resist this process, and that this process is associated with increased expression of CB.

Interspecific differences in the expression of the AMPA-type glutamate receptors and parvalbumin in the nucleus of Edinger-Westphal of chicks and pigeons.

The distribution of AMPA-type glutamate receptor (GluR) subunits was studied in the Edinger-Westphal nucleus (EW) of chicks and pigeons. GluR1, GluR2, GluR3 and GluR4 subunits appeared to be present in EW neurons of both species, but interspecific differences were observed in the abundance of the different types of subunits found in EW neurons. Of particular note, GluR2 immunoreactivity was present in the vast majority (ca. 80%) of neurons of pigeon EW but was found in only a small fraction (ca. 15%) of chick EW neurons. Scarcity of the GluR2 subunit in chick EW was confirmed by in situ hybridization. Because of the tendency for parvalbumin to be localized to neurons that are selectively deficient in GluR2, we also studied the localization of parvalbumin, as well as other calcium-binding proteins, in EW of chick and pigeon. Parvalbumin was found in more than 50% of chick EW neurons but was not detected in pigeon EW neurons. Our results suggest that there are major glutamatergic inputs to EW neurons in both pigeons and chicks. Furthermore, there are likely to be more AMPA-type calcium-permeable glutamate receptors in EW neurons of chick than in pigeon, since it is known that the subtype containing the edited GluR2 subunit is not calcium permeable.