RESUMEN
Cellular senescence is more than a proliferative arrest in response to various stimuli. Senescent cells (SC) participate in several physiological processes, and their adequate removal is essential to maintain tissue and organism homeostasis. However, SC accumulation in aging and age-related diseases alters the tissue microenvironment leading to deterioration. The immune system clears the SC, but the specific scenarios and mechanisms related to recognizing and eliminating them are unknown. Hence, we aimed to evaluate the existence of three regulatory signals of phagocytic function, CD47, major histocompatibility complex class I (MHC-I), and calreticulin, present in the membrane of SC. Therefore, primary fibroblasts were isolated from CD1 female mice lungs, and stress-induced premature senescence (SIPS) was induced with hydrogen peroxide. Replicative senescence (RS) was used as a second senescent model. Our results revealed a considerable increment of CD47 and MHC-I in RS and SIPS fibroblasts. At the same time, no significant changes were found in calreticulin, suggesting that those signals might be associated with evading immune system recognition and thus averting senescent cells clearance.
Asunto(s)
Antígenos CD1/metabolismo , Antígeno CD47/metabolismo , Senescencia Celular/fisiología , Fibroblastos/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Pulmón/metabolismo , Animales , Calbindina 2/metabolismo , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Fibroblastos/citología , Peróxido de Hidrógeno/toxicidad , Ratones , Cultivo Primario de CélulasRESUMEN
Depression is a neuropsychiatric disorder with a high impact on the worldwide population. To overcome depression, antidepressant drugs are the first line of treatment. However, pre-clinical studies have pointed out that antidepressants are not entirely efficacious and that the quality of the living environment after stress cessation may play a relevant role in increasing their efficacy. As it is unknown whether a short daily exposure to environmental enrichment during chronic stress and antidepressant treatment will be more effective than just the pharmacological treatment, this study analyzed the effects of fluoxetine, environmental enrichment, and their combination on depressive-associated behavior. Additionally, we investigated hippocampal neurogenesis in mice exposed to chronic mild stress. Our results indicate that fluoxetine reversed anhedonia. Besides, fluoxetine reversed the decrement of some events of the hippocampal neurogenic process caused by chronic mild stress. Conversely, short daily exposure to environmental enrichment changed the deterioration of the coat and anhedonia. Although, this environmental intervention did not produce significant changes in the neurogenic process affected by chronic mild stress, fluoxetine plus environmental enrichment showed similar effects to those caused by environmental enrichment to reverse depressive-like behaviors. Like fluoxetine, the combination reversed the declining number of Ki67, doublecortin, calretinin cells and mature newborn neurons. Finally, this study suggests that short daily exposure to environmental enrichment improves the effects of fluoxetine to reverse the deterioration of the coat and anhedonia in chronically stressed mice. In addition, the combination of fluoxetine with environmental enrichment produces more significant effects than those caused by fluoxetine alone on some events of the neurogenic process. Thus, environmental enrichment improves the benefits of pharmacological treatment by mechanisms that need to be clarified.
Asunto(s)
Anhedonia/efectos de los fármacos , Fluoxetina/farmacología , Hipocampo/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Estrés Psicológico/fisiopatología , Anhedonia/fisiología , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Calbindina 2/metabolismo , Proliferación Celular , Proteína Doblecortina/metabolismo , Ambiente , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Antígeno Ki-67/metabolismo , Ratones , Ratones Endogámicos BALB C , Estrés FisiológicoRESUMEN
GABAergic medium spiny neurons are the main neuronal population in the striatum. Calbindin is preferentially expressed in medium spiny neurons involved in the indirect pathway. The aim of the present work is to analyze the effect of perinatal asphyxia on different subpopulations of GABAergic neurons in the striatum and to assess the outcome of deep therapeutic hypothermia. The uterus of pregnant rats was removed by cesarean section and the fetuses were exposed to hypoxia by immersion in water (19 min) at 37°C (perinatal asphyxia). The hypothermic group was exposed to 10°C during 30 min after perinatal asphyxia. The rats were euthanized at the age of one month (adolescent/adult rats), their brains were dissected out and coronal sections were immunolabeled for calbindin, calretinin, NeuN, and reelin. Reelin+ cells showed no staining in the striatum besides subventricular zone. The perinatal asphyxia (PA) group showed a significant decrease in calbindin neurons and a paradoxical increase in neurons estimated by NeuN staining. Moreover, calretinin+ cells, a specific subpopulation of GABAergic neurons, showed an increase caused by PA. Deep hypothermia reversed most of these alterations probably by protecting calbindin neurons. Similarly, there was a reduction of the diameter of the anterior commissure produced by the asphyxia that was prevented by hypothermic treatment.
Asunto(s)
Asfixia Neonatal/terapia , Cuerpo Estriado/patología , Discinesias/prevención & control , Hipotermia Inducida/métodos , Trastornos Psicóticos/prevención & control , Animales , Animales Recién Nacidos , Comisura Anterior Cerebral/patología , Encéfalo/metabolismo , Encéfalo/patología , Calbindina 2/metabolismo , Calbindinas/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Cuerpo Estriado/metabolismo , Discinesias/etiología , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/patología , Masculino , Proteínas del Tejido Nervioso/metabolismo , Embarazo , Trastornos Psicóticos/etiología , Ratas , Ratas Sprague-Dawley , Proteína Reelina , Serina Endopeptidasas/metabolismoRESUMEN
The striatum is an essential component of the basal ganglia that regulatessensory processing, motor, cognition, and behavior. Depending on the species, the striatum shows a unique structure called caudate-putamen as in mice, or its separation into two regions called caudate and lenticular nuclei, the latter formed by putamen and globus pallidus areas, as in primates. These structures have two compartments, striosome and matrix. We investigated the structural organization, GABAergic and tyrosine hydroxylase (TH) expression in the striatum and globus pallidus of the South American plains vizcacha, Lagostomus maximus. Its striatum showed regionalization arising from the presence of an internal capsule, and a similar organization to a striosome-matrix compartmentalization. GABAergic neurons in the matrix of caudate exhibited parvalbumin, calretinin, calbindin, GAD65, and NADPH-d-immunoreactivity. These were also expressed in cells of the putamen with the exception of calretinin showing neurofibers localization. Globus pallidus showed parvalbumin- and GAD65-immunoreactive cells, and calretinin- and calbindin-immunoreactive neuropil, plus GABA-A-immunoreactive neurofibers. NADPH-d-, GAD65- and GABA-A-immunoreactive neurons were larger than parvalbumin-, calretinin-, and calbindin-immunoreactive cells, whereas calbindin-immunoreactive cells were the most abundant. In addition, TH-immunoreactive neuropil was observed in the matrix of the striatum. A significant larger TH-immunoreactive area and neuron number was found in females compared to males. The presence of an internal capsule suggests an adaptive advantage concerning motor and cognitive abilities favoring reaction time in response to predators. In an anatomy-evolutive perspective, the striatum of vizcacha seems to be closer to that of humans than to that of laboratory traditional models such as mouse.
Asunto(s)
Cuerpo Estriado/metabolismo , Neuronas GABAérgicas/metabolismo , Globo Pálido/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Animales , Calbindina 2/metabolismo , Calbindinas/metabolismo , Cuerpo Estriado/anatomía & histología , Femenino , Globo Pálido/anatomía & histología , Humanos , Inmunohistoquímica , Masculino , Ratones , Parvalbúminas/metabolismo , RoedoresRESUMEN
BACKGROUND: Hematoxylin-eosin (HE) staining of a full-thickness rectal wall fragment is classically used for the diagnosis of Hirschsprung disease (HD). However, this technique requires large fragments for a better diagnosis. Additionally, the histochemical and immunohistochemical methods of staining small fragments of rectal mucosal and submucosal biopsies are not available in all centers. Therefore, the possibility of diagnosing HD through HE staining in these biopsies could be a valuable alternative for centers that do not have more specific techniques. The objectives of the current investigation were to evaluate the concordance of the results obtained by HE staining and the calretinin method with acetylcholinesterase (AChE) activity in fragments of mucosa and submucosa in the diagnosis of HD. METHODS: For this study, 50 cases from our laboratory were selected. The tissue material was embedded in paraffin. Sixty levels of each fragment were utilized for HE, and the other 3 levels were used for calretinin. These slides were analyzed under the microscope, photographed and classified as either positive for HD when no ganglion cells were found with nerve trunks present or as negative when ganglion cells were found. The results from reading the slides were compared with those of AChE. RESULTS: Of the 50 cases evaluated by the HE technique, only 5 contradicted the diagnosis based on AChE, with a Kappa value of 0.800 and an accuracy of 90%. In the comparison between calretinin and AChE, 8 cases were discordant, with a Kappa value of 0.676 and an accuracy of 84%. CONCLUSIONS: The concordance of results from AChE and HE methods was satisfactory, allowing for the potential use of the HE method for fragments of mucosa and submucosa as a valid alternative in the diagnosis of HD. The immunohistochemical technique of calretinin did not show good agreement with the AChE activity in our study.
Asunto(s)
Calbindina 2/metabolismo , Enfermedad de Hirschsprung/patología , Mucosa Intestinal/patología , Recto/patología , Acetilcolinesterasa/metabolismo , Biopsia/métodos , Hematoxilina , Enfermedad de Hirschsprung/diagnóstico , Humanos , Inmunohistoquímica/métodos , Coloración y Etiquetado/métodosRESUMEN
The midline thalamus is reciprocally connected with the medial temporal lobe, where neural circuitry essential for spatial navigation and memory formation resides. Yet, little information is available on the dynamic relationship between activity patterns in the midline thalamus and medial temporal lobe. Here, we report on the functional heterogeneity of anatomically-identified thalamic neurons and the differential modulation of their activity with respect to dorsal hippocampal rhythms in the anesthetized mouse. Midline thalamic neurons expressing the calcium-binding protein calretinin, irrespective of their selective co-expression of calbindin, discharged at overall low levels, did not increase their activity during hippocampal theta oscillations, and their firing rates were inhibited during hippocampal sharp wave-ripples. Conversely, thalamic neurons lacking calretinin discharged at higher rates, increased their activity during hippocampal theta waves, but remained unaffected during sharp wave-ripples. Our results indicate that the midline thalamic system comprises at least two different classes of thalamic projection neuron, which can be partly defined by their differential engagement by hippocampal pathways during specific network oscillations that accompany distinct behavioral contexts. Thus, different midline thalamic neuronal populations might be selectively recruited to support distinct stages of memory processing, consistent with the thalamus being pivotal in the dialogue of cortical circuits.
Asunto(s)
Hipocampo/fisiología , Red Nerviosa/fisiología , Neuronas/fisiología , Lóbulo Temporal/fisiología , Tálamo/fisiología , Potenciales de Acción/fisiología , Animales , Calbindina 2/metabolismo , Calbindinas/metabolismo , Hipocampo/anatomía & histología , Memoria/fisiología , Ratones Endogámicos C57BL , Vías Nerviosas/fisiología , Neuronas/metabolismo , Lóbulo Temporal/anatomía & histología , Tálamo/anatomía & histologíaRESUMEN
The subdivisions of the medial geniculate complex can be distinguished based on the immunostaining of calcium-binding proteins and by the properties of the neurons within each subdivision. The possibility of changes in neurochemistry in this and other central auditory areas are important aspects to understand the basis that contributing to functional variations determined by environmental cycles or the animal's cycles of activity and rest. This study investigated, for the first time, day/night differences in the amounts of parvalbumin-, calretinin- and calbindin-containing neurons in the thalamic auditory center of a non-human primate, Sapajus apella. The immunoreactivity of the PV-IR, CB-IR and CR-IR neurons demonstrated different distribution patterns among the subdivisions of the medial geniculate. Moreover, a high number of CB- and CR-IR neurons were found during day, whereas PV-IR was predominant at night. We conclude that in addition to the chemical heterogeneity of the medial geniculate nucleus with respect to the expression of calcium-binding proteins, expression also varied relative to periods of light and darkness, which may be important for a possible functional adaptation of central auditory areas to environmental changes and thus ensure the survival and development of several related functions.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Cuerpos Geniculados/metabolismo , Animales , Vías Auditivas/citología , Vías Auditivas/metabolismo , Calbindina 2/metabolismo , Calbindinas/metabolismo , Cebus , Ritmo Circadiano , Cuerpos Geniculados/citología , Inmunohistoquímica , Técnicas In Vitro , Masculino , Neuronas/metabolismo , Parvalbúminas/metabolismo , Tálamo/metabolismoRESUMEN
The development of therapeutic approaches to improve the life quality of people suffering from different types of body paralysis is a current major medical challenge. Brain-machine interface (BMI) can potentially help reestablishing lost sensory and motor functions, allowing patients to use their own brain activity to restore sensorimotor control of paralyzed body parts. Chronic implants of multielectrodes, employed to record neural activity directly from the brain parenchyma, constitute the fundamental component of a BMI. However, before this technique may be effectively available to human clinical trials, it is essential to characterize its long-term impact on the nervous tissue in animal models. In the present study we evaluated how chronic implanted tungsten microelectrode arrays impact the distribution and morphology of interneurons reactive to calcium-binding proteins calbindin (CB), calretinin (CR) and parvalbumin (PV) across the rat's motor cortex. Our results revealed that chronic microelectrode arrays were well tolerated by the nervous tissue, with recordings remaining viable for up to 6 months after implantation. Furthermore, neither the morphology nor the distribution of inhibitory neurons were broadly impacted. Moreover, restricted microglial activation was observed on the implanted sites. On the whole, our results confirm and expand the notion that tungsten multielectrodes can be deemed as a feasible candidate to future human BMI studies.
Asunto(s)
Calbindina 1/metabolismo , Calbindina 2/metabolismo , Electrodos Implantados/efectos adversos , Implantes Experimentales/efectos adversos , Parvalbúminas/metabolismo , Animales , Ondas Encefálicas/fisiología , Interfaces Cerebro-Computador/efectos adversos , Masculino , Microglía/metabolismo , Corteza Motora/fisiología , Corteza Motora/cirugía , Ratas , Ratas WistarRESUMEN
BACKGROUND: Prefrontal cortex (PFC) represents the highest level of integration and control of psychic and behavioral states. Several dysfunctions such as autism, hyperactivity disorders, depression, and schizophrenia have been related with alterations in the prefrontal cortex (PFC). Among the cortical layers of the PFC, layer II shows a particular vertical pattern of organization, the highest cell density and the biggest non-pyramidal/pyramidal neuronal ratio. We currently characterized the layer II cytoarchitecture in human areas 10, 24, and 46. OBJECTIVE: We focused particularly on the inhibitory neurons taking into account that these cells are involved in sustained firing (SF) after stimuli disappearance. METHODS: Postmortem samples from five subjects who died by causes different to central nervous system diseases were studied. Immunohistochemistry for the neuronal markers, NeuN, parvalbumin (PV), calbindin (CB), and calretinin (CR) were used. NeuN targeted the total neuronal population while the rest of the markers specifically the interneurons. RESULTS: Cell density and soma size were statically different between areas 10, 46, 24 when using NeuN. Layer II of area 46 showed the highest cell density. Regarding interneurons, PV+-cells of area 46 showed the highest density and size, in accordance to the proposal of a dual origin of the cerebral cortex. Interhemispheric asymmetries were not identified between homologue areas. CONCLUSION: First, our findings suggest that layer II of area 46 exhibits the most powerful inhibitory system compared to the other prefrontal areas analyzed. This feature is not only characteristic of the PFC but also supports a particular role of layer II of area 46 in SF. Additionally, known functional asymmetries between hemispheres might not be supported by morphological asymmetries.
ANTECEDENTES: La corteza prefrontal (CPF) representa el nivel más alto de integración y control de funciones psíquicas y comportamentales. Varias patologías como autismo, desórdenes de hiperactividad, depresión y esquizofrenia se han relacionado con alteraciones de la CPF. La lámina II de las áreas que constituyen la CPF posee un patrón de organización vertical, una alta densidad celular y la mayor proporción de neuronas no-piramidal/piramidal. Sin embargo, la distribución del componente inhibitorio en estas regiones no se ha descrito. En el presente estudio nos propusimos caracterizar la lámina II de las áreas 10, 24 y 46 del humano, particularmente su componente inhibitorio teniendo en mente su participación en procesos de actividad sostenida relevantes cuando desaparece el estímulo. MÉTODOS: Se utilizaron muestras de cinco sujetos que fallecieron por causas diferentes a enfermedades del sistema nervioso. Se tomaron secciones de las áreas 10,24 y 46 de Brodmann y se procesaron con los anticuerpos contra NeuN para determinar la población neuronal total y contra parvalminina (PV), calbindina (CB) y calretinina )CR) para analizar la población de interneuronas. RESULTADOS: Los resultados no mostraron diferencias interhemisféricas entre las áreas. Sin embargo, las tres áreas seleccionadas son significativamente diferentes entre sí en todos los parámetros analizados. El área 46 posee la mayor densidad y tamaño de interneuronas positivas para PV. CONCLUSIONES: La ausencia de asimetrías morfológicas no permite explicar las asimetrías funcionales. La lámina II del área 46 posee el sistema inhibitorio más poderoso. Teniendo en cuenta la arquitectura modular de las capas supragranulares, este sistema inhibitorio subyace a la actividad sostenida, eje fundamental de la memoria operativa.
Asunto(s)
Interneuronas/citología , Neuronas/metabolismo , Corteza Prefrontal/citología , Adulto , Antígenos Nucleares/metabolismo , Calbindina 2/metabolismo , Calbindinas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Parvalbúminas/metabolismoRESUMEN
In the nervous system within physiological conditions, nitric oxide (NO) production depends on the activity of nitric oxide synthases (NOSs), and particularly on the expression of the neuronal isoform (nNOS). In the sensory systems, the role of NO is poorly understood. In this study, we identified nNOS-positive cells in the inner nuclear layer (INL) of the rat retina, with distinct characteristics such as somata size, immunolabeling level and location. Employing mathematical cluster analysis, we determined that nNOS amacrine cells are formed by two distinct populations. We next investigated the molecular identity of these cells, which did not show colocalization with calbindin (CB), choline acetyltransferase (ChAT), parvalbumin (PV) or protein kinase C (PKC), and only partial colocalization with calretinin (CR), revealing the accumulation of nNOS in specific amacrine cell populations. To access the functional, circuitry-related roles of these cells, we performed experiments after adaptation to different ambient light conditions. After 24h of dark-adaptation, we detected a subtle, yet statistically significant decrease in nNOS transcript levels, which returned to steady-state levels after 24h of normal light-dark cycle, revealing that nNOS expression is governed by ambient light conditions. Employing electron paramagnetic resonance (EPR), we demonstrated that dark-adaptation decreases NO production in the retina. Furthermore, nNOS accumulation changed in the dark-adapted retinas, with a general reduction in the inner plexiform layer. Finally, computational analysis based on clustering techniques revealed that dark-adaptation differently affected both types of nNOS-positive amacrine cells. Taken together, our data disclosed functional regulation of nNOS expression and activity, disclosing new circuitry-related roles of nNOS-positive cells. More importantly, this study indicated unsuspected roles for NO in the sensory systems, particularly related to adaptation to ambient demands.
Asunto(s)
Adaptación Ocular/fisiología , Regulación hacia Abajo/fisiología , Óxido Nítrico Sintasa/metabolismo , Retina/enzimología , Retina/fisiología , Animales , Calbindina 2/metabolismo , Calbindinas/metabolismo , Colina O-Acetiltransferasa/metabolismo , Análisis por Conglomerados , Espectroscopía de Resonancia por Spin del Electrón , Neuronas/metabolismo , Parvalbúminas/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Ratas Long-Evans , Retina/citologíaRESUMEN
Risperidone is an approved antipsychotic drug belonging to the chemical class of benzisoxazole. This drug has low solubility in aqueous medium and poor bioavailability due to extensive first-pass metabolism and high protein binding (>90%). Since new strategies to improve efficient treatments are needed, we studied the efficiency of anionic G4.5 PAMAM dendrimers as nanocarriers for this therapeutic drug. To this end, we explored dendrimer-risperidone complexation dependence on solvent concentration, pH and molar relationship. The best dendrimer-risperidone incorporation (46 risperidone molecules per dendrimer) was achieved with a mixture of chloroform:methanol 50â¶50 v/v solution pH 3. In addition, to explore the possible effects of this complex, in vivo studies were carried out in the zebrafish model. Changes in the development of dopaminergic neurons and motoneurons were studied using tyrosine hydroxylase and calretinin, respectively. Physiological changes were studied through histological sections stained with hematoxylin-eosin to observe possible morphological brain changes. The most significant changes were observed when larvae were treated with free risperidone, and no changes were observed when larvae were treated with the complex.
Asunto(s)
Antipsicóticos/farmacología , Dendrímeros/química , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Risperidona/farmacología , Animales , Antipsicóticos/química , Biomarcadores/metabolismo , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Calbindina 2/genética , Calbindina 2/metabolismo , Supervivencia Celular/efectos de los fármacos , Dendrímeros/farmacología , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/fisiología , Portadores de Fármacos , Expresión Génica , Concentración de Iones de Hidrógeno , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Risperidona/química , Solventes , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The suprachiasmatic nucleus (SCN), which is the main circadian biological clock in mammals, is composed of multiple cells that function individually as independent oscillators to express the self-sustained mRNA and protein rhythms of the so-called clock genes. Knowledge regarding the presence and localization of the proteins and neuroactive substances of the SCN are essential for understanding this nucleus and for its successful manipulation. Although there have been advances in the investigation of the intrinsic organization of the SCN in rodents, little information is available in diurnal species, especially in primates. This study, which explores the pattern of expression and localization of PER2 protein in the SCN of capuchin monkey, evaluates aspects of the circadian system that are common to both primates and rodents. Here, we showed that PER2 protein immunoreactivity is higher during the light phase. Additionally, the complex organization of cells that express vasopressin, vasoactive intestinal polypeptide, neuron-specific nuclear protein, calbindin and calretinin in the SCN, as demonstrated by their immunoreactivity, reveals an intricate network that may be related to the similarities and differences reported between rodents and primates in the literature.
Asunto(s)
Expresión Génica/fisiología , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/metabolismo , Animales , Arginina Vasopresina/metabolismo , Calbindina 2/metabolismo , Calbindinas/metabolismo , Cebus , Ritmo Circadiano/genética , Masculino , Proteínas del Tejido Nervioso/metabolismo , Proteínas Circadianas Period/genética , Estimulación Luminosa , ARN Mensajero/metabolismo , Serotonina/metabolismo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
The pathological evaluation of rectal biopsies for the diagnosis of Hirschsprung disease has been a challenging issue. We analyzed prospectively the usefulness of calretinin immunostaining and acetylcholinesterase (AChE) histochesmistry in rectal biopsies for the diagnosis of Hirschsprung disease. Frozen tissue samples from 43 patients were used for AChE histochemistry and paraffin-embedded sections for calretinin immunohistochemistry and conventional histology (hematoxylin and eosin [H&E]). Activity for AChE, was demonstrated in 13 of 43 cases, and the absence of immunoreactivity for calretinin was observed in 14 of 43 cases. Conventional histology (H&E) did not reveal ganglion cells in 24 of 43 cases. The results on calretinin were in good agreement with AChE according to the κ index (0.946; P<.001) and presented significantly higher specificity (96.7×63.3; P<.002) and accuracy (97.6×74.4; P<.003) when compared with conventional histology (H&E). The final diagnosis of Hirschsprung disease was confirmed in 13 of 43 patients who were submitted to surgical treatment. The results of the present study indicate that calretinin can be a good tool in ruling out the diagnosis of Hirschsprung disease, by showing positive staining in ganglion cells and intrinsic nerve fibers, whereas AChE is useful in confirming the diagnosis of Hirschsprung disease, by revealing activity of this enzyme in hypertrophied nerve fibers. The association between calretinin and AChE can be a useful panel for the histopathologic evaluation of rectal biopsies for the diagnosis of Hirschsprung disease.
Asunto(s)
Acetilcolinesterasa/metabolismo , Calbindina 2/metabolismo , Enfermedad de Hirschsprung/patología , Recto/patología , Biopsia , Brasil , Niño , Preescolar , Colectomía , Femenino , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/cirugía , Humanos , Lactante , Recién Nacido , Masculino , Estudios Prospectivos , Recto/metabolismo , Recto/cirugía , Sensibilidad y EspecificidadRESUMEN
The colocalization, number, and size of various classes of enteric neurons immunoreactive (IR) for the purinergic P2X2 and P2X7 receptors (P2X2R, P2X7R) were analyzed in the myenteric and submucosal plexuses of control, undernourished, and re-fed rats. Pregnant rats were exposed to undernourishment (protein-deprivation) or fed a control diet, and their offspring comprised the following experimental groups: rats exposed to a normal diet throughout gestation until postnatal day (P)42, rats protein-deprived throughout gestation and until P42, and rats protein-deprived throughout gestation until P21 and then given a normal diet until P42. Immunohistochemistry was performed on the myenteric and submucosal plexuses to evaluate immunoreactivity for P2X2R, P2X7R, nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, and calretinin. Double-immunohistochemistry of the myenteric and submucosal plexuses demonstrated that 100% of NOS-IR, calbindin-IR, calretinin-IR, and ChAT-IR neurons in all groups also expressed P2X2R and P2X7R. Neuronal density increased in the myenteric and submucosal plexuses of undernourished rats compared with controls. The average size (profile area) of some types of neurons in the myenteric and submucosal plexuses was smaller in the undernourished than in the control animals. These changes appeared to be reversible, as animals initially undernourished but then fed a normal diet at P21 (re-feeding) were similar to controls. Thus, P2X2R and P2X7R are present in NOS-positive inhibitory neurons, calbindin- and calretinin-positive intrinsic primary afferent neurons, cholinergic secretomotor neurons, and vasomotor neurons in rats. Alterations in these neurons during undernourishment are reversible following re-feeding.