Databases for technical aspects of immunohistochemistry: 2021 update.

With the aim of sharing information about the technical aspects of immunohistochemistry (IHC) and facilitating the selection of suitable antibodies for histopathological examination, this technical report describes the results of a questionnaire distributed during the period of 2018 to 2019 among members of the Conference on Experimental Animal Histopathology. Additionally, it describes the immunological properties and supplier details (clone, supplier, catalog number, species reactivity, etc.) as well as the IHC staining conditions (fixing solution, fixing time, embedding, antigen retrieval method, antibody dilution, incubation time, incubation temperature, positive control tissue, blocking condition, secondary antibody information, etc.) for a total of 509 primary antibodies (comprising 220 different types). These survey results were an update on the contents reported by CEAH in 2017.

Establishment of the Global SEND Alliance (G-SEND) in Japan and efficient creation of electronic SEND datasets between CROs.

The Standard for Exchange of Nonclinical Data (SEND), adopted by the US Food and Drug Administration (FDA), is a set of regulations for digitalization and standardization of nonclinical study data; thus, related organizations have begun implementing processes in support of SEND. The Global Editorial and Steering Committee (GESC), which provides oversight of the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND), has prepared the SEND Controlled Terminology (CT) for toxicologic pathology. SEND provides electronic data standards created by the Clinical Data Interchange Standards Consortium (CDISC), and CDISC also collaborates in the implementation of SEND. Furthermore, the Pharmaceutical Users Software Exchange (PhUSE), which includes members of the US FDA, has conducted various activities to promote realistic and effective methods to implement SEND. As we reported in 2015, there is a significant variation in the efficiency and quality of SEND data implementation across pharmaceutical companies and contractors (CROs) globally. To address this problem, the Global SEND Alliance (G-SEND) was established in August 2018 to facilitate the coordination and standardization of SEND datasets across CROs in Asia. This paper reports the first method for organizationally and jointly creating consistent SEND datasets between CROs using G-SEND.

Specific pathologist responses for Standard for Exchange of Nonclinical Data (SEND).

The Standard for Exchange of Nonclinical Data (SEND), introduced by the US Food and Drug Administration (FDA), is a scheme for the computerization, electronic application, and screening of preclinical data. Since its establishment, related organizations have been working together to implement SEND. However, it is difficult for individual pharmaceutical companies that often outsource to achieve complete compliance with SEND; hence, the cooperation of contract research organizations (CROs) and SEND Registered Solution Providers (RSPs) is indispensable. In SEND, most data, including those on pathology findings, are converted into controlled terminology (CT), but it is not a simple process to convert findings or levels of severity in the field of pathology, which is a descriptive science. The authors have successfully completed an FDA trial submission for a toxicology test conducted at a CRO and in doing so acquired important knowledge. This article presents a clear picture of such important knowledge from a pathologist's viewpoint.

Characteristics of corneal phospholipidosis induced by topical ocular application of chloroquine and amiodarone in rabbits.

Several cationic-amphiphilic drugs such as chloroquine and amiodarone are known to induce phospholipidosis in the cornea by systemic administration. However, the characteristics of ophthalmological and pathological changes when phospholipidosis-inducing drugs are topically applied have not been well studied. This study was conducted to investigate the characteristics of corneal changes caused by topical application of chloroquine and amiodarone to Japanese white rabbits. The changes were evaluated by ophthalmological, histopathological, and ultrastructural examinations. An in vivo confocal microscopy was also applied to the chloroquine-treated corneas. In both chloroquine- and amiodarone-treated corneas, diffuse cloudiness was observed by slit-lamp biomicroscopy, and its transparency increased with duration of dosing. Confocal microscopy showed punctate dots in the corneal epithelium. Histopathologically, cytoplasmic vacuolation was found in the corneal epithelium and keratocytes in both chloroquine- and amiodarone-treated eyes. Furthermore, foamy cytoplasm of the corneal endothelium was observed in the chloroquine-treated eyes. Ultrastructural examination showed multi-lamellar inclusion bodies or membrane-like debris in the lysosome-like vacuoles in the cytoplasm of corneal cells, which is a characteristic of the lesions of phospholipidosis. These changes disappeared after a withdrawal period. Continuous dosing of chloroquine resulted in corneal erosion and focal corneal opacity as shown by gross observation and slit-lamp biomicroscopy. Confocal microscopy could detect the corneal changes prior to the appearance of these ophthalmological changes. The present study showed that phospholipidosis caused by ocular administration of chloroquine and amiodarone first induces reversible diffuse corneal cloudiness. Confocal microscopy is a useful method for monitoring induction of corneal phospholipidosis.

Evaluation of a repeated-dose liver micronucleus assay with 2,6-dinitrotoluene using young adult rats.

As part of a collaborative study by the Mammalian Mutagenicity Study Group of the Environmental Mutagen Society of Japan, we examined micronucleus induction in hepatocytes following oral administration of 2,6-dinitrotoluene (2,6-DNT) at 30, 40, and 50mg/kg/day for 14 days or at 20, 30, and 40mg/kg/day for 28 days to young adult male rats. This compound is known to be a rat liver carcinogen. The formation of micronucleated hepatocytes was confirmed to be dose-dependent with statistically significant increases observed in both treatments. In contrast, no statistically significant changes in the percentage of micronucleated immature erythrocytes were observed in any dose group in the bone marrow micronucleus assay. These results indicated that the repeated-dose liver micronucleus assay has the potential to detect genotoxic hepatocarcinogens and can be integrated into general toxicological studies.

Unchanged distribution density of anionic sites on the glomerular wall in rats with active heymann nephritis.

In various kinds of glomerulonephritis, alteration of anionic charge on the glomerular basement membrane (GBM) and podocytes has been controversial for more than decade. To elucidate the relation between glomerular protein leakage and anionic sites on the glomerular wall, we examined the distribution of anionic sites on the GBM and podocytes of rats with active Heymann nephritis (AHN). Urinalysis for protein levels was conducted, and the kidneys were examined using electron microscopic cytochemistry for the assessment of anionic charge with two cationic probes. The anionic sites on podocytes were decreased in number in the AHN rats; however, the distributions of anionic sites on the GBM were similar in density to those seen in the control animals. From these results, we consider that the decrease in anionic charge density on podocytes might be attributable to protein leakage and that the charge barrier of the GBM is irrelevant to the protein leakage in AHN rats.

Unchanged distribution density of anionic sites on the glomerular wall in rats with streptozotocin-induced diabetic nephropathy.

As a cause of proteinuria in diabetic nephropathy, a decrease in anionic charge on the glomerular basement membrane (GBM) is considered to be related to protein leakage. However, the constancy of the anionic charge has been reported in several types of nephropathy. To elucidate the relation between glomerular protein leakage and anionic charge, we examined the distribution of anionic sites on the GBM and podocytes in diabetic rats induced by a single intravenous injection of 60 mg/kg of streptozotocin (STZ). Five months after the treatment with STZ, urinalysis for glucose and protein levels was conducted, and the kidneys were examined using electron microscopic cytochemistry for the assessment of anionic charge with two cationic probes. The distributions of anionic sites on the GBM demonstrated by two kinds of cationic markers in the diabetic rats were similar in density to those seen in the control animals. The distributions of anionic sites on the foot processes and cell membrane of podocytes were regular and also similar in density to that of the control group. From these results, we consider that the charge barrier of the GBM and podocytes is irrelevant to the protein leakage in diabetic rats.

Genotoxicity studies of 2,6-dinitrotoluene (2,6-DNT).

The potential genotoxicity of the rodent liver carcinogen 2,6-dinitrotoluene (2,6-DNT) was evaluated in compliance with the guidelines for genotoxicity studies of drugs (Notification No. 1604, Nov. 1, 1999, Ministry of Health and Welfare, Japan) and the OECD guidelines for the testing of chemicals by performing the bacterial reverse mutation (Ames) assay, the in vitro chromosomal aberration assay, and the in vivo comet assay (alkaline single cell gel electrophoresis) in rat liver. In the Ames assay, 2,6-DNT was moderately positive irrespective of metabolic activation. In the in vitro chromosomal aberration assay, under conditions where the test substance would precipitate out, weak structural aberrations were observed with or without S9 mix at each dose at which the cell growth rate was about 40 to 50%. The in vivo comet assay yielded positive results in rat liver; that is to say, the increases in % tail DNA in liver in the 25 and 50 mg/kg groups were observed statistically significantly and dose-dependent. Our findings are in accordance with previous findings in the in vivo/in vitro unscheduled DNA synthesis (UDS) assay in rat liver and in a young rat liver micronucleus assay, although the rat bone marrow micronucleus assay gave negative results. These results suggest that test systems using liver are a useful method for the in vivo genotoxicity assessment of chemicals that require metabolic activation.

Spontaneous vacuolar degeneration of the thyroid follicular epithelium in cynomolgus monkeys.

Vacuolar degeneration of the thyroid follicular epithelium was observed in two untreated female cynomolgus monkeys assigned to control groups. In light microscopy, large vacuoles containing a homogenous substance occupied the basal region of the epithelium, and the nuclei had shifted toward the apical region. The vacuoles showed negative reactions to PAS and thyroglobulin. Electron microscopic observation revealed dilatation of the rough endoplasmic reticulum corresponding to the vacuoles. The plasma TSH, T3 and T4 levels determined for the samples kept frozen were within the normal ranges, suggesting that the thyroid function was kept intact.

Pregnancy-associated changes in responsiveness of the porcine myometrium to bioactive substances.

To determine the pregnancy-associated changes in the porcine uterine contractility, the spontaneous contraction and the mechanical responses to bioactive substances of uteri in nonpregnant proestrus and pregnant pigs (25-60 days of gestation) were compared in vitro. Longitudinal muscle (LM) and circular muscle (CM) of the uterus exhibited spontaneous contraction, but the frequency in pregnant pigs was lower than that in the nonpregnant pigs. The duration and force of spontaneous contraction in the pregnant pigs were long and large compared with both in the nonpregnant pigs. L-Nitroarginine methylester (L-NAME) and 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,5-tetrahydro-benzo[cd]indol-2(1H)-one (DR4004) did not change the spontaneous contraction in the uteri of nonpregnant pigs but increased its amplitude in the uteri of pregnant pigs. Isoprenaline inhibited the uterine spontaneous contraction of the nonpregnant and pregnant pigs, and the inhibition was stronger in the pregnant than in the nonpregnant pigs. 5-Hydroxytryptamine also caused inhibition of spontaneous contraction in the uteri of nonpregnant pigs (CM>LM). In the pregnant pigs, sensitivity to 5-hydroxytryptamine increased in a muscle layer-dependent manner (LM>CM) and difference in the responsiveness between LM and CM decreased. Acetylcholine contracted the uterine LM and CM strips of the pregnant and nonpregnant pigs. The responsiveness of CM increased slightly during pregnancy, but that of the LM did not change. 5-Bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine (UK14304) caused contraction of only LM in the uteri of nonpregnant pigs, but contracted both LM and CM strips in the pregnant pigs. Oxytocin and prostaglandin F(2 alpha) also contracted the uteri of nonpregnant pigs (LM>CM). Pregnancy increased the contraction of both agents in the LM and CM, but the increment was marked in the CM. The contractile forces induced by all stimulants were increased (by 1.7- to 2.5-fold) in the LM and CM of pregnant pigs. In conclusion, (1) low frequency, slow kinetics and large force of spontaneous contraction are characteristics of the pregnant porcine uteri, and nitric oxide and 5-hydroxytryptamine are supposed to be partially involved in the regulation of spontaneous contraction, and (2) responses to both contractile and inhibitory agents are increased in the pregnant pigs. Increment of the responsiveness is conspicuous in the muscle layer that is less sensitive to each agonist in the uteri of nonpregnant pigs. According to the pregnancy-associated changes, muscle layer-related differences of responsiveness to bioactive substances in the nonpregnant pigs tend to decrease in the pregnant pigs.