Notes on the combined use of V-VIP and DAB peroxidase substrates for the detection of colocalising antigens

The purpose of the present report was to investigate to what extent the new peroxidase substrate Vector VIP (V-VIP) can be used in combination with DAB chromogen for the unequivocal and permanent detection of colocalising antigens within a single neurone, according to a two-colour paradigm. With this aim, retrograde tract-tracing with cholera toxin B subunit (CTB) or fluoro-gold (FG) was performed to disclose individual, identified subpopulations of neurones in the primate substantia nigra projecting to the caudate nucleus or to the putamen, respectively. Each tracer was detected by means of a PAP procedure and finally stained brown using DAB as a chromogen. Subsequently, both series of sections were processed for the immunocytochemical detection of tyrosine hydroxylase (TH). TH-immunoreactive neurones were stained purple with the peroxidase substrate V-VIP. As a result of the present procedure, several cell bodies of projection neurones, stained brown, can easily be identified within the primate substantia nigra. Some of these neurones additionally displayed purple TH immunoreaction product located in the neuronal dendrites. By contrast, CTB- or FG-unlabelled neurones only show the typical purple precipitate that belongs to V-VIP substrate, both in the cell body as well as in the dendrites

Relationships between thalamostriatal neurons and pedunculopontine projections to the thalamus: a neuroanatomical tract-tracing study in the rat

The present study aimed to investigate whether the pedunculopontine projection to the thalamus overlaps with identified thalamostriatal neurons. These projections were studied using a dual tract-tracing procedure combining anterogradely transported biotinylated dextran amine (pedunculopontine projections) and retrogradely transported Fluoro-Gold (thalamostriatal projections). Overlapping thalamic territories between thalamostriatal neurons and the axon terminals arising from the pedunculopontine tegmental nucleus were observed in the midline (paraventricular) and in the intralaminar (centrolateral, central medial, paracentral and parafascicular) thalamic nuclei. Other thalamic nuclei, such as the ethmoid, intermediodorsal, mediodorsal, paratenial, posteromedian, ventromedian, ventrolateral and rhomboid thalamic nuclei, displayed a lesser degree of overlap. These observations suggest the existence of presumptive contacts between thalamostriatal neurons and axons emerging from the pedunculopontine tegmental nucleus, therefore supporting the possible existence of feedback circuits in the rat basal ganglia in which the tegmento-thalamic projection would play a major role

Re-examination of the thalamostriatal projections in the rat with retrograde tracers

Topographical arrangements of thalamostriatal projection neurons was examined in the rat by the retrograde tract-tracing method. After injecting Fluoro-Gold (FG) and/or cholera toxin beta-subunit (CTB) in different regions of the caudate-putamen (CPu), distribution of retrogradely labeled neurons was observed in the thalamus. The main findings were as follows: (1) Retrogradely labeled neurons were seen in the midline-intralaminar thalamic nuclei in all rats examined in the present study. Neurons in the ventral lateral and posterior thalamic nuclear groups were also labeled in the rats which were injected with the tracer into the dorsal part of Cpu, but not in the rats which were injected with the tracer into the nucleus accumbens (Acb) and its adjavent regions in the ventromedial part of the Cpu. (2) Topographical organization was observed in the projections from the midline-intralaminar thalamic nuclei to the CPu. After the tracer injection into the dorsal part of the CPu or the ventral part of the CPu (including the Acb), labeled neurons in the midline-intralaminar thalamic nuclei were distributed predominantly in the lateral part of the intralaminar nuclei or the midline nuclei, respectively. On the other hand, after the tracer injection into the medial or the lateral part of the CPu, labeled neurons in the midline-intralaminar nuclei were distributed mainly in the dorsal or the ventral part of these nuclei, respectively. (3) Topographical organization was also observed in the thalamostriatal projections from the ventral and Pos. After the tracer injection into the rostral part of the CPu, labeled neurons were distributed mainly in the rostral part of the ventral nuclear group. On the other hand, after the tracer injection into the caudal part of the CPu, labeled neurons were distributed mainly in the caudal part of the ventral nuclear group, as well as in the posterior nuclear group

Multiple neuroanatomical tracing in primates

The present report deals with a multiple tract-tracing procedure in non-human primates enabling the simultaneous visualization of retrogradely transported Fluoro-Gold (FG) and cholera toxin B subunit (CTB) in combination with anterogradely transported biotinylated dextran amine (BDA). Two issues have played key roles on the achievement of this reliable procedure: first, the recent development of a commercial antiserum against FG that allows us to convert the original fluorescent signal of this dye in a permanent precipitate via standard peroxidase-anti-peroxidase methods; second, the introduction of the novel peroxidase substrate Vector(R) VIP (V-VIP), resulting in a purple precipitate. The combination of these neuroanatomical tracers in one and the same histological section opens a possibility for the permanent visualization of the convergence of inputs from a particular brain area onto identified, two different subsets of projection cells of another area. Furthermore, this combination of three tracers emerges as a powerful technical tool for obtaining broad amounts of complementary data regarding the monkey brain connectivity, thus significantly reducing the number of animals needed to complete a particular study

Striatal input from the ventrobasal complex of the rat thalamus

We have analyzed whether caudal regions of the caudate putamen receive direct projections from thalamic sensory relay nuclei such as the ventrobasal complex. To this aim, the delivery of the retrograde neuroanatomical tracer Fluoro-Gold into the caudal caudate putamen resulted in the appearance of retrogradely labeled neurons in the ventral posteromedial and ventral posterolateral thalamic nuclei. These projections were further confirmed with injections of the anterograde tracers biotinylated dextran amine or Phaseolus vulgaris leucoagglutinin into these thalamic nuclei, by showing the existence of axonal terminal fields located in the caudal striatum. These results support the existence of direct projections linking the thalamic ventrobasal complex and the caudal striatum in the rat, probably via collateralization of thalamocortical axons when passing through the caudate putamen, and therefore supporting the putative involvement of the caudal striatum in sensory-related functions

Multiple axonal tracing: simultaneous detection of three tracers in the same section

Multiple neuroanatomical tract-tracing methods are important tools for elucidating the connectivity between different populations of neurons. Evaluation of the question as to whether two specific fiber inputs converge on a particular, identified population of projection neurons requires the application of a triple-staining procedure that allows the unequivocal detection of three markers in a single section. The present report deals with a combination of tracing methods using anterogradely transported Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine in conjunction with retrogradely transported Fluoro-Gold. These tracers were simultaneously detected according to a three-color paradigm, which includes the use of three different peroxidase substrates (nickel-enhanced diaminobenzidine, diaminobenzidine, and Vector VIP), thus resulting in three distinct precipitates: black, brown, and purple. We illustrate this method by showing convergence of projections arising from neurons located in two separate basal ganglia-related nuclei onto identified thalamostriatal projection neurons

Thalamic interaction between the input and the output systems of the basal ganglia

The striatal return through the thalamus is largely neglected in current studies dealing with basal ganglia function, and its role within this circuitry remains obscure. In this contribution the thalamus is regarded as an important place of interaction between the input and the output organization of the basal ganglia. In support of this idea, a brief overview is provided of some of the most recent findings concerning the thalamus in relation to the basal ganglia circuitry. In particular, we have focused on the thalamostriatal projections themselves, on the output of the basal ganglia to the thalamus and also on the overlapping territories between the thalamic projection of the output nuclei and the thalamostriatal neurons. These data support the existence of several thalamic feedback circuits within the basal ganglia neural system. Finally, some considerations are provided upon the functional significance of these thalamic feedback circuits in the overall organization of the basal ganglia

Coronavirus disease 2019 in chronic kidney disease

The clinical spectrum of coronavirus disease 2019 (COVID-19) infection ranges from asymptomatic infection to severe pneumonia with respiratory failure and even death. More severe cases with higher mortality have been reported in older patients and in those with chronic illness such as hypertension, diabetes or cardiovascular diseases. In this regard, patients with chronic kidney disease (CKD) have a higher rate of all-type infections and cardiovascular disease than the general population. A markedly altered immune system and immunosuppressed state may predispose CKD patients to infectious complications. Likewise, they have a state of chronic systemic inflammation that may increase their morbidity and mortality. In this review we discuss the chronic immunologic changes observed in CKD patients, the risk of COVID-19 infections and the clinical implications for and specific COVID-19 therapy in CKD patients. Indeed, the risk for severe COVID-19 is 3-fold higher in CKD than in non-CKD patients; CKD is 12-fold more frequent in intensive care unit than in non-hospitalized COVID-19 patients, and this ratio is higher than for diabetes or cardiovascular disease; and acute COVID-19 mortality is 15-25% for haemodialysis patients even when not developing pneumonia

REVASCULARIZAÇÃO CONVENCIONAL ULTRA-DISTAL NA ISQUEMIA CRÍTICA

Introdução: A cirurgia convencional de revascularização ultra-distal continua a ser pouco utilizada pela maioria dos cirurgiões vasculares. No entanto, os escassos estudos publicados mostram resultados muito favoráveis em termos de sobrevida e salvação de membro. Objetivos: Avaliar os resultados da cirurgia convencional de revascularização ultra-distal em doentes com isquemia crítica grau IV (estadios 5 e 6 da classificação de Rutherford); analisar os detalhes técnicos e de decisão cirúrgica que influenciam os resultados obtidos. Material e métodos: Estudo retrospetivo, envolvendo todos os doentes admitidos nesta instituição com isquemia crítica nos estadios 5 e 6 da classificação de Rutherford, submetidos a cirurgia convencional de revascularização ultra-distal. Foi considerado critério de inclusão a cirurgia de bypass às artérias plantares comum, interna ou externa e à artéria pediosa. Foi avaliada a mortalidade, taxa de preservação de membro, sobrevida livre de amputação major e permeabilidade da revascularização, por intermédio de curvas de Kaplan-Meier. Resultados: Entre Abril de 2012 e Março de 2016, 41 doentes (34 homens e 7 mulheres) com uma média de idades de 69.1 anos, foram submetidos a 50 procedimentos de revascularização ultra-distal, 35 dos quais à artéria pediosa, 10 a artérias plantares e 5 a procedimentos de resgate de failing graft. Em 84% das revascularizações (n=42) utilizou-se substituto venoso autólogo, sendo os restantes bypass compostos de PTFE e veia (n=8). O follow-up médio foi de 20.8 meses (2-47 meses). Não registámos qualquer mortalidade aos 30 dias e durante o follow-up a mortalidade global foi 7.3% (n=3). Ocorreram 9 oclusões das revascularizações (2 precoces e 7 tardias) e 5 amputações major. A permeabilidade primária foi de 78% e a permeabilidade primária assistida de 82%. A taxa de preservação de membro foi de 88.9% e, no final do follow-up, a sobrevida livre de amputação foi de 66%. Conclusões: Os resultados demonstram que, em centros especializados e com elevado volume, esta técnica é muito eficaz na salvação de membros em isquemia crítica nos estadios 5 e 6 da classificação de Rutherford. Estes procedimentos devem ser tomados em consideração na ausência de outros vasos pontáveis ou como resgate, em membros já submetidos a procedimentos de revascularização prévios

Benthic community recovery from brine impact after the implementation of mitigation measures

In many regions, seawater desalination is a growing industry that has its impact on benthic communities. This study analyses the effect on benthic communities of a mitigation measure applied to a brine discharge, using polychaete assemblages as indicator. An eight-year study was conducted at San Pedro del Pinatar (SE Spain) establishing a grid of 12 sites at a depth range of 29–38 m during autumn. Brine discharge started in 2006 and produced a significant decrease in abundance, richness and diversity of polychaete families at the location closest to the discharge, where salinity reached 49. In 2010, a diffuser was deployed at the end of the pipeline in order to increase the mixing, to reduce the impact on benthic communities. After implementation of this mitigation measure, the salinity measured close to discharge was less than 38.5 and a significant recovery in polychaete richness and diversity was detected, to levels similar to those before the discharge. A less evident recovery in abundance was also observed, probably due to different recovery rates of polychaete families. Some families like Paraonidae and Magelonidae were more tolerant to this impact. Others like Syllidae and Capitellidae recovered quickly, although still affected by the discharge, while some families such as Sabellidae and Cirratulidae appeared to recover more slowly.These surveys were funded by the Mancomunidad de Canales del Taibilla