Vancouver call for action to strengthen expertise in radiological protection worldwide.

Ionising radiation has been used for over a century for peaceful purposes, revolutionising health care and promoting well-being through its application in industry, science, and medicine. For almost as long, the International Commission on Radiological Protection (ICRP) has promoted understanding of health and environmental risks of ionising radiation and developed a protection system that enables the safe use of ionising radiation in justified and beneficial practices, providing protection from all sources of radiation. However, we are concerned that a shortage of investment in training, education, research, and infrastructure seen in many sectors and countries may compromise society's ability to properly manage radiation risks, leading to unjustified exposure to or unwarranted fear of radiation, impacting the physical, mental, and social well-being of our peoples. This could unduly limit the potential for research and development in new radiation technologies (healthcare, energy, and the environment) for beneficial purposes. ICRP therefore calls for action to strengthen expertise in radiological protection worldwide through: (1) National governments and funding agencies strengthening resources for radiological protection research allocated by governments and international organisations, (2) National research laboratories and other institutions launching and sustaining long-term research programmes, (3) Universities developing undergraduate and graduate university programmes and making students aware of job opportunities in radiation-related fields, (4) Using plain language when interacting with the public and decision makers about radiological protection, and (5) Fostering general awareness of proper uses of radiation and radiological protection through education and training of information multipliers. The draft call was discussed with international organisations in formal relations with ICRP in October 2022 at the European Radiation Protection Week in Estoril, Portugal, and the final call announced at the 6th International Symposium on the System of Radiological Protection of ICRP in November 2022 in Vancouver, Canada.

Perspectives on tolerability and reasonableness.

Judgements on tolerability and reasonableness are central to the optimisation of protection. There are currently several international developments regarding these key considerations which will contribute to the review and evolution of the system of radiological protection. The IRPA15 International Congress brought together the principal issues currently under discussion, and the outcome of these discussions is presented.

Summary of the 2021 ICRP workshop on the future of radiological protection.

The International Commission on Radiological Protection (ICRP) has embarked on a process to review and revise the current System of Radiological Protection ('the System'). To stimulate discussion, the ICRP published two open-access articles: one on aspects of the System that might require review, and another on research that might improve the scientific foundation of the System. Building on these articles, the ICRP organized a Workshop on the Future of Radiological Protection as an opportunity to engage in the review and revision of the System. This digital workshop took place from 14 October-3 November 2021 and included 20 live-streamed and 43 on-demand presentations. Approximately 1500 individuals from 100 countries participated. Based on the subjects covered by the presentations, this summary is organized into four broad areas: the scientific basis, concepts and application of the System; and the role of the ICRP. Some of the key topics that emerged included the following: classification of radiation-induced effects; adverse outcome pathway methodologies; better understanding of the dose-response relationship; holistic and reasonable approaches to optimization of protection; radiological protection of the environment; ethical basis of the System; clarity, consistency and communication of the System; application of the System in medicine and application of the principles of justification and optimization of protection.

Areas of research to support the system of radiological protection.

This document presents the ICRP's updated vision on "Areas of Research to Support the System of Radiological Protection", which have been previously published in 2017. It aims to complement the research priorities promoted by other relevant international organisations, with the specificity of placing them in the perspective of the evolution of the System of Radiological Protection. This document contributes to the process launched by ICRP to review and revise the System of Radiological Protection that will update the 2007 General Recommendations in ICRP Publication 103.

Keeping the ICRP recommendations fit for purpose.

The International Commission on Radiological Protection (ICRP) has embarked on a review and revision of the system of Radiological Protection that will update the 2007 general recommendations in ICRPPublication 103. This is the beginning of a process that will take several years, involving open and transparent engagement with organisations and individuals around the world. While the system is robust and has performed well, it must adapt to address changes in science and society to remain fit for purpose. The aim of this paper is to encourage discussions on which areas of the system might gain the greatest benefit from review, and to initiate collaborative efforts. Increased clarity and consistency are high priorities. The better the system is understood, the more effectively it can be applied, resulting in improved protection and increased harmonisation. Many areas are identified for potential review including: classification of effects, with particular focus on tissue reactions; reformulation of detriment, potentially including non-cancer diseases; re-evaluation of the relationship between detriment and effective dose, and the possibility of defining detriments for males and females of different ages; individual variation in the response to radiation exposure; heritable effects; and effects and risks in non-human biota and ecosystems. Some of the basic concepts are also being considered, including the framework for bringing together protection of people and the environment, incremental improvements to the fundamental principles of justification and optimisation, a broader approach to protection of individuals, and clarification of the exposure situations introduced in 2007. In addition, ICRP is considering identifying where explicit incorporation of the ethical basis of the system would be beneficial, how to better reflect the importance of communications and stakeholder involvement, and further advice on education and training. ICRP invites responses on these and other areas relating to the review of the System of Radiological Protection.

Translating science into recommendations: the development of NCRP Report No. 180.

The development of new recommendations for radiological protection is a complex process of translating scientific knowledge of radiation risks into a coherent system for protection, using experience and ethical values. From 2014 to 2019, the US National Council on Radiation Protection and Measurements (NCRP) engaged in the development of updated recommendations to replace those previously published in 1993. This process included considering the experiences gained with implementation of the NCRP recommendations, and the recommendations of the International Commission on Radiological Protection. The new recommendations, now published as NCRP Report No. 180, go beyond the previous recommendations of the ICRP and NCRP, providing new approaches to emergency response and protection of the environment, covering additional topics not previously addressed in medical exposure. The system of protection continues to rely on justification of actions, optimisation of protection, and use of individual dose criteria to achieve the best protection for each exposure situation. For the first time in general recommendations, ethical values are discussed, and their use in complex decision making to achieve sustainable and supportable decisions outlined. The importance of stakeholder engagement is highlighted, as is the relationship with a radiation protection and safety culture. This paper outlines some of the questions and considerations that were part of the development of NCRP Report No. 180.

NCRP Report no.180-management of exposure to ionizing radiation: NCRP radiation protection guidance for the United States.

NCRP Report No. 180, 'Management of Exposure to Ionizing Radiation: Radiation Protection Guidance for the United States (2018)' was developed by Council Committee 1. The report builds and expands upon previous recommendations of NCRP and ICRP, covering exposure to radiation and radioactive materials for five exposure categories: occupational, public, medical, emergency workers, and nonhuman biota. Actions to add, increase, reduce or remove a source of exposure to humans require justification. Optimisation of protection universally applies, taking into account societal, economic, and environmental factors; addressing all hazards, and striving for continuous improvement when it is reasonable to do so. Numeric protection criteria for management of dose to an individual for a given exposure situation are provided, and differ in some respects from ICRP. A specific numeric criterion is suitable to be designated as a regulatory dose limit only when the source of exposure is stable, characterised, and the responsible organisation has established an appropriate radiation control program in advance of source introduction. Medical exposure includes patients, comforters and caregivers of a patient, and voluntary participants in biomedical research. Emergency workers are a new exposure category; their exposure is treated separately from occupational, public or medical exposure, and numeric criteria are provided for deterministic and stochastic effects. For nonhuman biota, the focus is on population maintenance of the affected species, and a guideline is provided for when additional assessment may be necessary. In addition, the recommendations emphasise that: ethical principles support decision-making; stakeholder engagement is necessary in deciding suitable management of their radiation exposure; and a strong safety culture is intrinsic to effective radiation protection programs.

Artificial intelligence and amniotic fluid multiomics: prediction of perinatal outcome in asymptomatic women with short cervix.

OBJECTIVE: To evaluate the application of artificial intelligence (AI), i.e. deep learning and other machine-learning techniques, to amniotic fluid (AF) metabolomics and proteomics, alone and in combination with sonographic, clinical and demographic factors, in the prediction of perinatal outcome in asymptomatic pregnant women with short cervical length (CL). METHODS: AF samples, which had been obtained in the second trimester from asymptomatic women with short CL (< 15 mm) identified on transvaginal ultrasound, were analyzed. CL, funneling and the presence of AF 'sludge' were assessed in all cases close to the time of amniocentesis. A combination of liquid chromatography coupled with mass spectrometry and proton nuclear magnetic resonance spectroscopy-based metabolomics, as well as targeted proteomics analysis, including chemokines, cytokines and growth factors, was performed on the AF samples. To determine the robustness of the markers, we used six different machine-learning techniques, including deep learning, to predict preterm delivery < 34 weeks, latency period prior to delivery < 28 days after amniocentesis and requirement for admission to a neonatal intensive care unit (NICU). Omics biomarkers were evaluated alone and in combination with standard sonographic, clinical and demographic factors to predict outcome. Predictive accuracy was assessed using the area under the receiver-operating characteristics curve (AUC) with 95% CI, sensitivity and specificity. RESULTS: Of the 32 patients included in the study, complete omics, demographic and clinical data and outcome information were available for 26. Of these, 11 (42.3%) patients delivered ≥ 34 weeks, while 15 (57.7%) delivered < 34 weeks. There was no statistically significant difference in CL between these two groups (mean ± SD, 11.2 ± 4.4 mm vs 8.9 ± 5.3 mm, P = 0.31). Using combined omics, demographic and clinical data, deep learning displayed good to excellent performance, with an AUC (95% CI) of 0.890 (0.810-0.970) for delivery < 34 weeks' gestation, 0.890 (0.790-0.990) for delivery < 28 days post-amniocentesis and 0.792 (0.689-0.894) for NICU admission. These values were higher overall than for the other five machine-learning methods, although each individual machine-learning technique yielded statistically significant prediction of the different perinatal outcomes. CONCLUSIONS: This is the first study to report use of AI with AF proteomics and metabolomics and ultrasound assessment in pregnancy. Machine learning, particularly deep learning, achieved good to excellent prediction of perinatal outcome in asymptomatic pregnant women with short CL in the second trimester. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

The new mandate and work of ICRP Committee 4.

Committee 4 of the International Commission on Radiological Protection (ICRP) is charged with the development of principles and recommendations on radiological protection of people and the environment in all exposure situations. For the term beginning in July 2017, the Committee has a total of 18 members from 12 countries. The programme of work includes a wide range of activities in five major thematic areas. The first is the consolidation and preparation of reports elaborating application of the system of protection in existing exposure situations. Second is the continuation of work on emergency exposure situations, and ICRP updates to recommendations in light of the accident at Fukushima Daiichi nuclear power plant. Third is examination of fundamentals of protection recommendations, including the ethical principles underlying the recommendations and application of those principles in practical decision making. Fourth is the new area of integration of protection of the environment into the system of protection. Finally, Committee 4 continues work to prepare specific topical reports on subjects in which additional information is useful to understand and apply the Commission's recommendations in particular circumstances.

Overview of ICRP Committee 4: application of the Commission's recommendations.

Committee 4 develops principles and recommendations on radiological protection of people in all exposure situations. The committee meeting in 2014 was hosted by GE Healthcare in Arlington Heights, IL, USA on 27 July-1 August 2014. The programme of work of Committee 4 encompasses several broad areas, including a series of reports covering various aspects of existing exposure situations, leading the efforts of the International Commission on Radiological Protection (ICRP) to update and elaborate recommendations in light of the accident at Fukushima Daiichi nuclear power plant for emergencies and living in contaminated areas, elaborating the underpinnings of the system of radiological protection, and developing focussed reports on specific topic areas in consultation with ICRP's special liaison organisations. Committee 4 has six active Task Groups working on naturally occurring radioactive material; cosmic radiation in aviation; updates of ICRP Publications 109 and 111; ethics of radiological protection; surface and near-surface disposal of solid radioactive waste; and exposures resulting from contaminated sites from past industrial, military, and nuclear activities. In addition, there is a Working Party on tolerability of risk, and ongoing work with the various special liaison organisations of ICRP.

Review of the ICRP system of protection: the approach to existing exposure situations.

The International Commission on Radiological Protection (ICRP) system of protection consists of existing, planned, and emergency exposure situations. With the 2007 Recommendations in ICRP Publication 103, a coherent approach has been established that emphasises the optimisation of protection with appropriate constraints or reference levels in each exposure situation. Existing exposure situations pose unique challenges because the source of exposure already exists, and it may not always be possible to control the source directly. This is the case for naturally occurring sources, which are ubiquitous in the environment and vary widely in the magnitude of exposures that may be received by individuals. Decisions on protection strategies must consider a graded, pragmatic, and flexible approach for dealing with exposure of members of the public, and those that may be occupationally exposed while working with naturally occurring sources. Although limits are not applicable, aspects of the management approach for planned exposure situations may be appropriate, depending upon the magnitude of exposures.

ICRP Publication 124: Protection of the Environment under Different Exposure Situations.

In this report, the Commission describes its framework for protection of the environment and how it should be applied within the Commission's system of protection. The report expands upon its objectives in relation to protection of the environment, in so far as it relates to the protection of animals and plants (biota) in their natural environment, and how these can be met by the use of Reference Animals and Plants (RAPs); their Derived Consideration Reference Levels (DCRLs), which relate radiation effects to doses over and above their normal local background natural radiation levels; and different potential pathways of exposure. The report explains the different types of exposure situations to which its recommendations apply; the key principles that are relevant to protection of the environment; and hence how reference values based on the use of DCRLs can be used to inform on the appropriate level of effort relevant to different exposure situations. Further recommendations are made with regard to how the Commission's recommendations can be implemented to satisfy different forms of environmental protection objectives, which may require the use of representative organisms specific to a site, and how these may be compared with the reference values. Additional information is also given with regard to, in particular, communication with other interested parties and stakeholders. Issues that may arise in relation to compliance are also discussed, and the final chapter discusses the overall implications of the Commission's work in this area to date. Appendices A and B provide some numerical information relating to the RAPs. Annex C considers various existing types of environmental protection legislation currently in place in relation to large industrial sites and practices, and the various ways in which wildlife are protected from various threats arising from such sites.

ICRP Publication 125: Radiological Protection in Security Screening.

The use of technologies to provide security screening for individuals and objects has been increasing rapidly, in keeping with the significant increase in security concerns worldwide. Within the spectrum of technologies, the use of ionizing radiation to provide backscatter and transmission screening capabilities has also increased. The Commission has previously made a number of statements related to the general topic of deliberate exposures of individuals in non-medical settings. This report provides advice on how the radiological protection principles recommended by the Commission should be applied within the context of security screening. More specifically, the principles of justification, optimisation of protection, and dose limitation for planned exposure situations are directly applicable to the use of ionising radiation in security screening. In addition, several specific topics are considered in this report, including the situation in which individuals may be exposed because they are concealed ('stowaways') in a cargo container or conveyance that may be subject to screening. The Commission continues to recommend that careful justification of screening should be considered before decisions are made to employ the technology. If a decision is made that its use is justified, the framework for protection as a planned exposure situation should be employed, including optimization of protection with the use of dose constraints and the appropriate provisions for authorisation and inspection.

ICRP, 123. Assessment of radiation exposure of astronauts in space. ICRP Publication 123.

During their occupational activities in space, astronauts are exposed to ionising radiation from natural radiation sources present in this environment. They are, however, not usually classified as being occupationally exposed in the sense of the general ICRP system for radiation protection of workers applied on Earth. The exposure assessment and risk-related approach described in this report is clearly restricted to the special situation in space, and should not be applied to any other exposure situation on Earth. The report describes the terms and methods used to assess the radiation exposure of astronauts, and provides data for the assessment of organ doses. Chapter 1 describes the specific situation of astronauts in space, and the differences in the radiation fields compared with those on Earth. In Chapter 2, the radiation fields in space are described in detail, including galactic cosmic radiation, radiation from the Sun and its special solar particle events, and the radiation belts surrounding the Earth. Chapter 3 deals with the quantities used in radiological protection, describing the Publication 103 (ICRP, 2007) system of dose quantities, and subsequently presenting the special approach for applications in space; due to the strong contribution of heavy ions in the radiation field, radiation weighting is based on the radiation quality factor, Q, instead of the radiation weighting factor, wR. In Chapter 4, the methods of fluence and dose measurement in space are described, including instrumentation for fluence measurements, radiation spectrometry, and area and individual monitoring. The use of biomarkers for the assessment of mission doses is also described. The methods of determining quantities describing the radiation fields within a spacecraft are given in Chapter 5. Radiation transport calculations are the most important tool. Some physical data used in radiation transport codes are presented, and the various codes used for calculations in high-energy radiation fields in space are described. Results of calculations and measurements of radiation fields in spacecraft are given. Some data for shielding possibilities are also presented. Chapter 6 addresses methods of determining mean absorbed doses and dose equivalents in organs and tissues of the human body. Calculated conversion coefficients of fluence to mean absorbed dose in an organ or tissue are given for heavy ions up to Z=28 for energies from 10 MeV/u to 100 GeV/u. For the same set of ions and ion energies, mean quality factors in organs and tissues are presented using, on the one hand, the Q(L) function defined in Publication 60 (ICRP, 1991), and, on the other hand, a Q function proposed by the National Aeronautics and Space Administration. Doses in the body obtained by measurements are compared with results from calculations, and biodosimetric measurements for the assessment of mission doses are also presented. In Chapter 7, operational measures are considered for assessment of the exposure of astronauts during space missions. This includes preflight mission design, area and individual monitoring during flights in space, and dose recording. The importance of the magnitude of uncertainties in dose assessment is considered. Annex A shows conversion coefficients and mean quality factors for protons, charged pions, neutrons, alpha particles, and heavy ions(2 < Z ≤2 8), and particle energies up to 100 GeV/u.

Vulvodynia and fungal association: a preliminary report.

Vulvodynia (vulvar pain syndrome) is a chronic multifactorial disease affecting almost 13 million women in the USA and can lead to morbidity and a reduced quality of life. We hypothesize that an initial microbiological insult in the vagina causes modifications in the biological vaginal milieu and/or an alteration on the lactobacilli flora. The vaginal milieu responds to the insult by developing an inflammatory reaction with abnormal cytokine production. These hypotheses were tested quantifying vaginal lactobacillus and cytokines, in patients with vulvodynia compared to matched healthy controls. Our preliminary data suggest a vaginal flora alteration and an immunological response involving Candida in patients with vulvodynia. Ongoing studies will assist us to clarify these findings.

US NRC discussion of options to revise radiation protection recommendations.

The Nuclear Regulatory Commission (NRC) is continuing the process of engaging stakeholders on issues associated with possible changes to the radiation protection regulations contained in 10 CFR Part 20, and other parts of the NRC regulations, to increase alignment with international recommendations. The Commission is particularly seeking to explore implications, as appropriate and where scientifically justified, of greater alignment with the 2007 Recommendations of the International Commission for Radiological Protection. Other information from national and international sources is also being considered. Given that the NRC regulations provide adequate protection, the discussion has been focusing on discerning the benefits and burdens associated with revising the radiation protection regulatory framework. NRC, through three Federal Register Notices, has officially solicited comments on a series of key issues, and has conducted a series of facilitated workshops to encourage feedback from a wide range of stakeholders. The issues include the use of updated scientific methodologies and terminology, the occupational dose limits, and the use of the concepts of constraints in optimisation. NRC staff provided a policy paper with recommendations to the Commission on April 25, 2012 (NRC, 2012).

Assessment of image registration accuracy in three-dimensional transrectal ultrasound guided prostate biopsy.

PURPOSE: Prostate biopsy, performed using two-dimensional (2D) transrectal ultrasound (TRUS) guidance, is the clinical standard for a definitive diagnosis of prostate cancer. Histological analysis of the biopsies can reveal cancerous, noncancerous, or suspicious, possibly precancerous, tissue. During subsequent biopsy sessions, noncancerous regions should be avoided, and suspicious regions should be precisely rebiopsied, requiring accurate needle guidance. It is challenging to precisely guide a needle using 2D TRUS due to the limited anatomic information provided, and a three-dimensional (3D) record of biopsy locations for use in subsequent biopsy procedures cannot be collected. Our tracked, 3D TRUS-guided prostate biopsy system provides additional anatomic context and permits a 3D record of biopsies. However, targets determined based on a previous biopsy procedure must be transformed during the procedure to compensate for intraprocedure prostate shifting due to patient motion and prostate deformation due to transducer probe pressure. Thus, registration is a critically important step required to determine these transformations so that correspondence is maintained between the prebiopsied image and the real-time image. Registration must not only be performed accurately, but also quickly, since correction for prostate motion and deformation must be carried out during the biopsy procedure. The authors evaluated the accuracy, variability, and speed of several surface-based and image-based intrasession 3D-to-3D TRUS image registration techniques, for both rigid and nonrigid cases, to find the required transformations. METHODS: Our surface-based rigid and nonrigid registrations of the prostate were performed using the iterative-closest-point algorithm and a thin-plate spline algorithm, respectively. For image-based rigid registration, the authors used a block matching approach, and for nonrigid registration, the authors define the moving image deformation using a regular, 3D grid of B-spline control points. The authors measured the target registration error (TRE) as the postregistration misalignment of 60 manually marked, corresponding intrinsic fiducials. The authors also measured the fiducial localization error (FLE), the effect of segmentation variability, and the effect of fiducial distance from the transducer probe tip. Lastly, the authors performed 3D principal component analysis (PCA) on the x, y, and z components of the TREs to examine the 95% confidence ellipsoids describing the errors for each registration method. RESULTS: Using surface-based registration, the authors found mean TREs of 2.13 +/- 0.80 and 2.09 +/- 0.77 mm for rigid and nonrigid techniques, respectively. Using image-based rigid and non-rigid registration, the authors found mean TREs of 1.74 +/- 0.84 and 1.50 +/- 0.83 mm, respectively. Our FLE was 0.21 mm and did not dominate the overall TRE. However, segmentation variability contributed substantially approximately50%) to the TRE of the surface-based techniques. PCA showed that the 95% confidence ellipsoid encompassing fiducial distances between the source and target registra- tion images was reduced from 3.05 to 0.14 cm3, and 0.05 cm3 for the surface-based and image-based techniques, respectively. The run times for both registration methods were comparable at less than 60 s. CONCLUSIONS: Our results compare favorably with a clinical need for a TRE of less than 2.5 mm, and suggest that image-based registration is superior to surface-based registration for 3D TRUS-guided prostate biopsies, since it does not require segmentation.

ICRP Publication 111 - Application of the Commission's recommendations to the protection of people living in long-term contaminated areas after a nuclear accident or a radiation emergency.

In this report, the Commission provides guidance for the protection of people living in long-term contaminated areas resulting from either a nuclear accident or a radiation emergency. The report considers the effects of such events on the affected population. This includes the pathways of human exposure, the types of exposed populations, and the characteristics of exposures. Although the focus is on radiation protection considerations, the report also recognises the complexity of post-accident situations, which cannot be managed without addressing all the affected domains of daily life, i.e. environmental, health, economic, social, psychological, cultural, ethical, political, etc. The report explains how the 2007 Recommendations apply to this type of existing exposure situation, including consideration of the justification and optimisation of protection strategies, and the introduction and application of a reference level to drive the optimisation process. The report also considers practical aspects of the implementation of protection strategies, both by authorities and the affected population. It emphasises the effectiveness of directly involving the affected population and local professionals in the management of the situation, and the responsibility of authorities at both national and local levels to create the conditions and provide the means favouring the involvement and empowerment of the population. The role of radiation monitoring, health surveillance, and the management of contaminated foodstuffs and other commodities is described in this perspective. The Annex summarises past experience of longterm contaminated areas resulting from radiation emergencies and nuclear accidents, including radiological criteria followed in carrying out remediation measures.

Signal transduction mechanisms of K+-Cl- cotransport regulation and relationship to disease.

The K+-Cl- cotransport (COT) regulatory pathways recently uncovered in our laboratory and their implication in disease state are reviewed. Three mechanisms of K+-Cl- COT regulation can be identified in vascular cells: (1) the Li+-sensitive pathway, (2) the platelet-derived growth factor (PDGF)-sensitive pathway and (3) the nitric oxide (NO)-dependent pathway. Ion fluxes, Western blotting, semi-quantitative RT-PCR, immunofluorescence and confocal microscopy were used. Li+, used in the treatment of manic depression, stimulates volume-sensitive K+-Cl- COT of low K+ sheep red blood cells at cellular concentrations <1 mM and inhibits at >3 mM, causes cell swelling, and appears to regulate K+-Cl- COT through a protein kinase C-dependent pathway. PDGF, a potent serum mitogen for vascular smooth muscle cells (VSMCs), regulates membrane transport and is involved in atherosclerosis. PDGF stimulates VSM K+-Cl- COT in a time- and concentration-dependent manner, both acutely and chronically, through the PDGF receptor. The acute effect occurs at the post-translational level whereas the chronic effect may involve regulation through gene expression. Regulation by PDGF involves the signalling molecules phosphoinositides 3-kinase and protein phosphatase-1. Finally, the NO/cGMP/protein kinase G pathway, involved in vasodilation and hence cardiovascular disease, regulates K+-Cl- COT in VSMCs at the mRNA expression and transport levels. A complex and diverse array of mechanisms and effectors regulate K+-Cl- COT and thus cell volume homeostasis, setting the stage for abnormalities at the genetic and/or regulatory level thus effecting or being affected by various pathological conditions.

Quantitative in situ hybridization for peptide mRNAs in mouse brain.

The objective was to determine the feasibility of using a radioactive capture method (Fuji FLA 2000) and image analysis system for the measurement of peptide mRNA levels in specific brain regions in mice. As a test mRNA, we chose vasopressin (VP) and oxytocin (OT) because they are expressed in abundance in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). A comparison was made between free-floating and slide-mounted sections to determine which method yielded better results. Mouse brains were fixed in 4% paraformaldehyde (PFA) and processed for in situ hybridization using 35S-oligonucleotide probes for VP and OT. After overnight hybridization and high stringency washes, 25-microm brain sections and 14C standards were exposed to a BAS-IIIs Fuji imaging film over a range of times (4 h-6 days). Results showed that there was an intense hybridization reaction in the PVN and SON, making it possible to distinguish the specific brain regions. Using Image Gauge Software, the signal was quantified in PVN and SON. A comparison of the different exposure times showed that the signal could be measured after as little as 4 h. The intensity readings increased over time while the calculated radioactivity remained constant. The free-floating method was superior to the slide-based system, providing a lower background and a higher signal. The data illustrates the applicability of the phosphor imaging system for the reproducible measurement of mRNA levels in discrete regions of the mouse brain.