RESUMEN
Genomic technologies are increasingly used clinically for both diagnosis and guiding cancer therapy. However, formalin fixation can compromise DNA quality. This study aimed to optimise tissue fixation using normal colon, liver and uterus (n=8 each) by varying neutral buffered formalin (NBF) concentration (1%-5% w/v) and fixation time (24-48 hours). Fixation using 4% NBF improved DNA quality (assessed by qPCR) compared with routine (4% unbuffered formal saline-fixed) specimens (p<0.01). Further improvements were achieved by reducing NBF concentration (p<0.00001), whereas fixation time had no effect (p=0.110). No adverse effects were detected by histopathological or QuPath morphometric analysis. Immunohistochemistry for multicytokeratin and α-smooth muscle actin revealed no changes in staining specificity or intensity in any tissue other than on liver multicytokeratin staining intensity, where the effect of fixation time was more significant (p=0.0004) than NBF concentration (p=0.048). Thus, reducing NBF concentration can maximise DNA quality without compromising tissue morphology or standard histopathological analyses.
Asunto(s)
ADN/aislamiento & purificación , Fijadores/farmacología , Formaldehído/farmacología , Adhesión en Parafina/normas , Enfermedades del Colon/patología , Femenino , Humanos , Inmunohistoquímica/normas , Hepatopatías/patología , Mejoramiento de la Calidad , Coloración y Etiquetado/normas , Fijación del Tejido/normas , Enfermedades Uterinas/patologíaRESUMEN
AIMS: Histochemical staining of tissue is a fundamental technique in tissue diagnosis and research, but it suffers from significant variability. Efforts to address this include laboratory quality controls and quality assurance schemes, but these rely on subjective interpretation of stain quality, are laborious and have low reproducibility. We aimed (1) to develop a method for histochemical stain quantification using whole slide imaging and image analysis and (2) to demonstrate its usefulness in measuring staining variation. METHODS: A method to quantify the individual stain components of histochemical stains on virtual slides was developed. It was evaluated for repeatability and reproducibility, then applied to control sections of an appendix to quantify H&E staining (H/E intensities and H:E ratio) between automated staining machines and to measure differences between six regional diagnostic laboratories. RESULTS: The method was validated with <0.5% variation in H:E ratio measurement when using the same scanner for a batch of slides (ie, it was repeatable) but was not highly reproducible between scanners or over time, where variation of 7% was found. Application of the method showed H:E ratios between three staining machines varied from 0.69 to 0.93, H:E ratio variation over time was observed. Interlaboratory comparison demonstrated differences in H:E ratio between regional laboratories from 0.57 to 0.89. CONCLUSIONS: A simple method using whole slide imaging can be used to quantify and compare histochemical staining. This method could be deployed in routine quality assurance and quality control. Work is needed on whole slide imaging devices to improve reproducibility.
Asunto(s)
Apéndice/patología , Técnicas de Laboratorio Clínico/normas , Interpretación de Imagen Asistida por Computador/normas , Laboratorios/normas , Ensayos de Aptitud de Laboratorios/normas , Indicadores de Calidad de la Atención de Salud/normas , Coloración y Etiquetado/normas , Automatización de Laboratorios , Colorantes/normas , Eosina Amarillenta-(YS)/normas , Hematoxilina/normas , Humanos , Variaciones Dependientes del Observador , Valor Predictivo de las Pruebas , Control de Calidad , Reproducibilidad de los Resultados , Manejo de Especímenes/normasRESUMEN
PURPOSE: Intracranial norepinephrine release has been associated with post-carotid endarterectomy hypertension in human beings. To study this phenomenon under more controlled conditions, we studied the relationship of cerebral catecholamines and blood pressure in gerbils, whose cerebral circulation is similar to that in human beings. METHODS: Twelve anesthetized gerbils underwent iliac artery blood pressure monitoring and in vivo electrochemistry catecholamine monitoring with use of catecholamine-specific electrodes placed stereotactically into the cerebral striatum. Six gerbils underwent 10 minutes of bilateral carotid artery occlusion (ischemic), whereas six underwent carotid artery dissection without occlusion (control). RESULTS: The control group demonstrated a continuous gradual decline in blood pressure and striatal catecholamine during the 150-minute observation period. In contrast the ischemic gerbils demonstrated a sharp catecholamine rise during ischemia, a marked catecholamine drop shortly after carotid artery unclamping, and then a secondary larger catecholamine release that peaks in 60 minutes and gradually returns to baseline in 120 minutes. The blood pressure closely followed the catecholamine levels, with a sharp 20 mm Hg rise in blood pressure above baseline during carotid artery occlusion, followed by a dramatic 10 mm Hg drop below baseline after carotid artery unclamping and then a gradual rise of the blood pressure 25 mm Hg above baseline, which peaks in 80 minutes, with a gradual decline to the same blood pressure as in the control subjects 120 minutes after ischemia. CONCLUSION: We conclude that striatal catecholamine release correlates with peripheral blood pressure during transient cerebral ischemia and reperfusion. This phenomenon may explain the mechanism of post-carotid endarterectomy hypertension in human beings, and this gerbil model can be used to study its prevention and treatment.
Asunto(s)
Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Dopamina/metabolismo , Gerbillinae/fisiología , Hipertensión/fisiopatología , Ataque Isquémico Transitorio/fisiopatología , Animales , Determinación de la Presión Sanguínea/métodos , Endarterectomía Carotidea , Femenino , Hipertensión/etiología , Ataque Isquémico Transitorio/complicaciones , Masculino , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/fisiopatología , Factores de TiempoRESUMEN
Male and female Mongolian gerbils (Meriones unguiculatus) carrying three coat color alleles, agouti, sandy, and black, were tested for 15 min under three cage temperature conditions, warm (35-40 degrees C), neutral (20 degrees C), and cold (0-5 degrees C). A frequency count of scratching, face and body wash, belly/side rubs, and shaking was taken of these animals' activities during the test sessions. Sex differences were not evident but coat color and age temperature affected the belly/side rubs and shaking activities. There were also effects of cage temperature but not coat color on face/body wash and scratching. It appears that animals with these coat color variants manifest disparate patterns of COBS behavior when encountering different thermal environments.
Asunto(s)
Regulación de la Temperatura Corporal/genética , Gerbillinae/genética , Aseo Animal/fisiología , Color del Cabello/genética , Fenotipo , Animales , Femenino , Lípidos/fisiología , MasculinoRESUMEN
The effects of the opiate antagonist, naloxone, on seizure tendencies of the black or nonagouti line of Mongolian gerbils (Meriones unguiculatus) carrying the a/a coat color allele was investigated. The animals were tested under the following conditions: mock infection; 0.9% NaCl injection; and naloxone at doses of 1, 2, 5, and 10 mg/kg body weight. The results indicated that when naloxone was at doses of 2 mg/kg body weight and higher, there was an increase in the animals' seizure latencies relative to that manifested under the basal condition.
Asunto(s)
Gerbillinae/genética , Naloxona/farmacología , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/genética , Convulsiones/genética , Convulsiones/fisiopatología , Animales , Encéfalo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electroencefalografía/efectos de los fármacos , Tiempo de Reacción/fisiología , betaendorfina/genética , betaendorfina/fisiologíaRESUMEN
To clarify the nature of ischemic striatal dopamine release during the earliest periods of neuronal injury, we used chronoamperometry to measure dopamine levels every 60 seconds during various durations of ischemia in 32 gerbils. Catecholamine-selective electrodes were implanted into the brains of anesthetized gerbils subjected to 2, 5, or 10 minutes of transient forebrain ischemia or permanent forebrain ischemia. Four control animals showed a stable chronoamperometric baseline. In the six gerbils subjected to permanent ischemia, dopamine release was rapid during early ischemia and slowed with time. The four animals subjected to 2 minutes of ischemia showed minimal dopamine release. The six gerbils subjected to 5 minutes of ischemia demonstrated a noticeable dopamine release during ischemia, and three of the six developed a massive secondary dopamine release during reperfusion. All six animals subjected to 10 minutes of ischemia demonstrated a similar biphasic dopamine release twice the size of that observed in the 5-minute group. Pretreatment with pargyline in six additional gerbils subjected to 10 minutes of ischemia failed to modify significantly this biphasic pattern of dopamine release. We conclude that dopamine release occurs very early during ischemia and that its magnitude correlates with the duration of an ischemia insult. Reperfusion is associated with an even larger striatal dopamine release. This previously unreported biphasic dopamine release phenomenon may have important clinical implications in the management of cerebral ischemia.
Asunto(s)
Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Ataque Isquémico Transitorio/metabolismo , Animales , Circulación Cerebrovascular , Electroquímica , Femenino , Gerbillinae , Ataque Isquémico Transitorio/fisiopatología , Neuronas , ReperfusiónRESUMEN
Female gerbils (Meriones unguiculatus) of three distinct coat colors (agouti, black, and sandy or pink-eyed dilution) were tested in a Y-maze whose arms led to compartments containing unfamiliar male gerbils of varying coat colors. The stimulus animals were separated from the females by a Plexiglas door. The trials lasted for 2 min and each female was exposed to the following four combinations: two males of the same coloring as the female; one male of the same color and another of a different color from the female; both males of different color from the female. The number of crossings to the left and right arms was relayed by photocells to an IBM PC computer. The results indicate that agouti females preferred visiting the arm occupied by agouti males while those of the other coat colors showed no preference for the "wild-type" males. Instead, sandy and black female gerbils preferred to be in proximity with those of non-wild types.
Asunto(s)
Gerbillinae/fisiología , Color del Cabello/fisiología , Conducta Sexual Animal/fisiología , Especificidad de la Especie , Animales , Nivel de Alerta/fisiología , Conducta de Elección/fisiología , Aprendizaje Discriminativo/fisiología , Femenino , Masculino , Orientación/fisiologíaRESUMEN
Seizure tendencies of three separate lines of Mongolian gerbils Meriones unguiculatus carrying three different coat color alleles were investigated. These alleles were agouti (A/-), black or nonagouti (a/a), or sandy (pink-eyed dilution p/p). Each animal was stroked on the back and then placed in a novel cage for 5 min while its seizure activity was measured in terms of latency, duration, and severity (grade). The results indicate that gerbils which are homozygous recessive at the pink-eyed dilution locus (sandy) exhibit less severe and shorter seizures than others. However, gerbils which are homozygous recessive at the agouti gene locus (black) show a shorter latency to manifest seizures than the other animals. These results indicate that the genetic mechanism determining coat color in Mongolian gerbils may also influence the susceptibility of these animals to seizure arising from novel stimulation.