Secretory factors released from high dose radiation-activated osteoclasts increase the expression level of pain-associated neuropeptides in sensory neuronal cultures.

Stereotactic Body Radiation Therapy for lung tumors near the chest wall often causes significant chest wall pain (CWP), negatively impacting patients' quality of life. The mechanisms behind SBRT-induced CWP remain unclear and may involve multiple factors. We investigated the potential crosstalk between radiation-activated osteoclasts and sensory neurons, focusing on osteoclast-derived factors in CWP. Using the murine pre-osteoclast cell line Raw264.7, we induced differentiation with RANKL, followed by 10Gy gamma-irradiation. Conditioned media from these irradiated osteoclasts was used to treat sensory neuronal cultures from mouse dorsal root ganglia. Neuronal cultures were also directly exposed to 10Gy radiation, with and without osteoclast co-culture. Analysis of osteoclast markers and pain-associated neuropeptides was conducted using RT-qPCR and histochemical staining. Osteoclast differentiation and activity were inhibited using Osteoprotegerin and risedronate. Results showed that high-dose radiation significantly increased osteoclast size, resorption pit size, and activity biomarkers. Neurons treated with CM from irradiated osteoclasts showed increased expression of pain-associated neuropeptides CGRP and Substance P, which was mitigated by osteoprotegerin and risedronate. This study suggests that high-dose radiation enhances osteoclast activity, upregulating pain-associated neuropeptides in sensory neurons, and that inhibitors like osteoprotegerin and risedronate may offer therapeutic strategies for managing radiation-induced pain.

Expression of genes associated with apoptosis in the residual yolk sac during the perihatch period of broiler chicks.

The yolk sac (YS) consists of the yolk and the surrounding YS tissue, which provides essential nutrients and physiological functions for the developing embryo. After the YS is internalized into the abdominal cavity of the embryonic chick, the YS starts to degrade. Apoptosis, or programmed cell-death, is speculated to be the mechanism behind degradation of the YS. The objective of this study was to determine if degradation of the YS tissue was mediated by apoptosis during the perihatch period. The YS tissue was collected from broiler chicks from embryonic d 17 to d 7 posthatch. The mRNA abundance of genes that are involved in the regulation, initiation, and execution of apoptosis were analyzed by qPCR. The mRNA for Bcl2, Bcl2L11, cytochrome C and caspases 3, 6, 7, 8, 9, and 18 all showed a linear response from embryonic d 17 to d 7 posthatch. To confirm the role of apoptosis in the degradation of the YS tissue, a DNA fragmentation assay was performed. Degradation of genomic DNA in the YS tissue started on day of hatch. The characteristic ladder of oligonucleosomal-sized DNA fragments was observed on d 3, 5, and 7 posthatch. The combined gene expression and DNA fragmentation results demonstrate that degradation of the YS posthatch is mediated by apoptosis.

Runting Stunting Syndrome in Broiler Chickens Is Associated with Altered Intestinal Stem Cell Morphology and Gene Expression.

Runting stunting syndrome (RSS) in broiler chickens is characterized by altered intestinal morphology and gene expression and stunted growth. The objective of this study was to conduct a retrospective study of gene expression in stem and differentiated cells in the small intestine of RSS chicks. Two different models of RSS were analyzed: broiler chicks that were experimentally infected and broiler chicks that were naturally infected. Experimentally infected chicks were exposed to litter from infected flocks (RSS-litter chicks) or infected with astrovirus (RSS-astrovirus chicks). Intestinal samples from naturally infected chicks showing clinical signs of RSS were acquired from commercial farms in Georgia and were brought into a poultry diagnostic lab (RSS-clinical-GA) and from farms in Brazil that had a history of RSS (RSS-clinical-BR). The RSS-clinical-BR chicks were separated into those that were positive or negative for gallivirus based on DNA sequencing. Intestinal morphology and intestinal cell type were identified in archived formalin-fixed, paraffin-embedded tissues. In situ hybridization for cell-specific mRNA was used to identify intestinal stem cells expressing olfactomedin 4 (Olfm4), proliferating cells expressing Ki67, absorptive cells expressing sodium glucose cotransporter 1 (SGLT1) and peptide transporter 1 (PepT1), and goblet cells expressing mucin 2 (Muc2). RSS-litter and RSS-clinical-GA chicks showed 4% to 7.5% cystic crypts, while gallivirus-positive RSS-clinical-BR chicks showed 11.7% cystic crypts. RSS-astrovirus and gallivirus-negative RSS-clinical-BR chicks showed few cystic crypts. RSS-litter and gallivirus-positive RSS-clinical-BR chicks showed an increase in crypt depth compared to control or gallivirus-negative chicks, respectively. There was no expression of Olfm4 mRNA in the stem cells of RSS-litter and RSS-clinical-GA chicks, in contrast to the normal expression of Olfm4 mRNA in RSS-astrovirus and RSS-clinical-BR chicks. All chicks regardless of infection status showed normal expression of Ki67 mRNA in crypt cells, Muc2 mRNA in goblet cells, and SGLT1 or PepT1 mRNA in enterocytes. These results demonstrate that RSS, which can be induced by different etiologies, can show differences in the expression of the stem cell marker Olfm4.

El síndrome del enanismo infeccioso en pollos de engorde se asocia con alteración de la morfología de las células madre intestinales y la expresión de genes. El síndrome del enanismo infeccioso (con las siglas en inglés RSS) en pollos de engorde se caracteriza por alteraciones en la morfología intestinal y en la expresión de genes, además de retraso en el crecimiento. El objetivo de este estudio fue realizar un estudio retrospectivo de la expresión genética en células madre y células diferenciadas en el intestino delgado de pollitos con el síndrome del enanismo infeccioso. Se analizaron dos modelos diferentes del síndrome del enanismo infeccioso: en pollos de engorde que fueron infectados experimentalmente y en pollos de engorde infectados naturalmente. Los pollitos infectados experimentalmente se expusieron a la cama de parvadas infectadas (RSS-litter chicks), o infectados con astrovirus (RSS-astrovirus chicks). Se adquirieron muestras intestinales de pollitos infectados naturalmente que mostraban signos clínicos del síndrome del enanismo infeccioso de granjas comerciales en Georgia y se llevaron a un laboratorio de diagnóstico avícola (RSS-Clinical-GA) y de granjas en Brasil que tenían antecedentes del síndrome del enanismo infeccioso (RSS-Clinical-BR). Los pollitos de granjas de Brasil (RSS-Clinical-BR) se separaron en aquellos que fueron positivos o negativos para gallivirus de acuerdo con la secuenciación del ADN. Se identificaron la morfología intestinal y el tipo de células intestinales en tejidos archivados fijados con formalina e incluidos en parafina. La hibridación in situ para ARNm específico de células se utilizó para identificar células madre intestinales que expresan olfactomedina 4 (Olfm4), células en proliferación que expresaban Ki67, células absorbentes que expresan el cotransportador 1 de glucosa y sodio (SGLT1) y el transportador de péptidos 1 (PepT1), y células caliciformes que expresan mucina 2 (Muc2). Los pollos expuestos a cama infectada (RSS-litter) y los infectados naturalmente de Georgia (RSS-clinical-GA) mostraron entre un 4% y un 7.5% de criptas quísticas, mientras que los pollos infectados de granjas de Brasil (RSS-clinical-BR) que eran positivos para gallivirus mostraron un 11.7% de criptas quísticas. Los pollos infectados con astrovirus (RSS-astrovirus chicks) y los pollos de Brasil (RSS-clinical-BR) que eran negativos para gallivirus mostraron pocas criptas quísticas. Los pollos expuestos a cama infectada (RSS-litter chicks) y los pollos infectados de Brasil (RSS-clinical-BR) que eran positivos para gallivirus mostraron un aumento en la profundidad de las criptas en comparación con los pollos control o negativos para el gallivirus, respectivamente. No se observó expresión de ARNm de Olfm4 en las células madre de pollitos expuestos a cama infectada (RSS-litter chicks) ni en pollos infectados de Georgia (RSS-clinical-GA), en contraste con la expresión normal de ARNm de Olfm4 en pollitos infectados con astrovirus (RSS-astrovirus chicks) y en pollitos infectados de Brasil (RSS-clinical-BR). Todos los pollos, independientemente del estado de infección, mostraron una expresión normal de ARNm para Ki67 en las células de la cripta, de ARNm para Muc2 en las células caliciformes y ARNm de SGLT1 o PepT1 en los enterocitos. Estos resultados demuestran que el síndrome del enanismo infeccioso, que puede ser inducido por diferentes etiologías, puede mostrar diferencias en la expresión del marcador para células madre Olfm4.