Streptococcus pneumoniae is the leading cause of bacterial meningitis. Pneumococcal meningitis is associated with the highest mortality among bacterial meningitis and it may also lead to neurological sequelae despite the use of antibiotic therapy. Experimental animal models of pneumococcal meningitis are important to study the pathogenesis of meningitis, the host immune response induced after infection, and the efficacy of novel drugs and vaccines.S. pneumoniae were 3.2 × 10, 2.9 × 10 and 1.9 × 102 colony forming units, respectively. Characterisation of the disease caused by the type 4 strain showed that in moribund mice systemic dissemination of pneumococci to blood and spleen occurred. Histological analysis of the brain of animals infected with type 4 S. pneumoniae proved the induction of meningitis closely resembling the disease in humans.In the present work, we describe in detail a simple, reproducible and efficient method to induce pneumococcal meningitis in outbred mice by using the intracranial subarachnoidal route of infection. Bacteria were injected into the subarachnoid space through a soft point located 3.5 mm rostral from the bregma. The model was tested with several doses of pneumococci of three capsular serotypes , and mice survival was recorded. Lethal doses killing 50 % of animals infected with type 2, 3 and 4 The proposed method for inducing pneumococcal meningitis in outbred mice is easy-to-perform, fast, cost-effective, and reproducible, irrespective of the serotype of pneumococci used. Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae [S. pneumoniae in humans and especially in children [S. pneumoniae in mice may also occur through retrograde axonal transport along olfactory neurons [Bacterial meningitis is an important infection of the central nervous system (CNS), and the three major responsible bacteria are eumoniae . Despiteeumoniae ,3. Develchildren . The patchildren ,2. Howev neurons . Once th neurons ,2.S. pneumoniae infection, and (iv) test the efficacy of novel antibiotics and vaccine candidates. Both infant and adult rats [S. pneumoniae [S. pneumoniae in humans. However, those models present the disadvantage that PM is induced in about half of the animals, while the remaining mice may die of sepsis without developing meningitis. Models of meningitis induced by the i.p. route were employed to carry out therapeutic studies [et al. [et al. is an useful and reliable system for causing PM in the mouse [et al., who induced meningitis via inoculation of S. pneumoniae (type 3) into the cisterna magna of C57BL/6 mice and investigated the function of nitric oxide in the disease [Animal models of PM have been developed in order to: (i) characterise the pathogenesis of meningitis, (ii) analyse the role of pneumococcal virulence factors in the disease, (iii) understand the host immune response to ult rats -9, and ault rats -13 have eumoniae . Howevereumoniae -17, (ii)eumoniae ,19, or  as a model strain. The proposed method is precise, simple, cost-effective, fast and reproducible, and the disease induced closely resembles PM in humans.i.e. lethargy, paralysis) for several weeks after inoculation (data not shown).The bregma is the intersection of the coronal and sagittal sutures of the skull and can be recognised in mice by visual examination of the exposed skull Fig. . We inocThese data allowed localisation of the anatomical coordinates of the inoculation site and proved the suitability of the i.c. subarachnoidal infection technique.After characterisation of the inoculation site and technique, mice were infected with pneumococci, and the establishment of PM was evaluated and clearly evidenced by histological analysis (see below).50) were calculated. We chose three commonly used S. pneumoniae strains, such as D39, HB565, and TIGR4. The D39 strain (type 2) is the encapsulated parent of the rough type 2 R36A strain used by Avery [50 of 3.2 × 10 and 2.9 × 10 colony forming units (CFU), respectively (data not shown). The TIGR4 strain was less virulent in the i.c. subarachnoidal infection model compared to D39 and HB565, as its LD50 was 1.9 × 102 CFU (data not shown).In order to test the model with different pneumococcal serotypes, dose-dependent survival studies were performed, and the lethal doses killing 50% of animals , while it considerably decreased at the highest dose (105 CFU) leading to rapid death of all mice.To describe the features of PM in detail following i.c. subarachnoidal infection of mice, we chose to characterise PM induced by the TIGR4 genome strain. For this purpose, we performed time-dependent survival studies, bacterial counts in different organs/tissues, and histological analysis of brain and spleen (see below). In order to study animal survival, five groups of MF1 mice were infected with doses of TIGR4 increasing from 10 to 10rve Fig. . After i5 CFU of TIGR4 were sacrificed, samples collected and appropriately treated, and viable counts were carried out. Moribund animals showed comparable bacterial counts in the brain (3.1 × 106 ± 1.3 × 106 CFU/organ). Similarly, dissemination from the brain to vital organs occurred and was consistent in all animals, with bacterial counts of 3.8 × 106 ± 4.8 × 106 CFU and 2.1 × 108 ± 3.0 × 108 CFU in the spleen and blood, respectively (data not shown).To determine the number of pneumococci in brain, spleen and blood at the final stages of the disease, moribund mice infected with 10i.e. hunchbacked, photophobic, lethargic), but they did not develop hemiparesis or plegia. Moribund mice that had received 102, 103, 104, and 105 CFU were humanely killed at 72, 72, 48, and 24 hours post-infection, respectively. Two animals for each pneumococcal dose and three additional control mice were sacrificed. Brains and spleens were excised and treated for both haematoxilin-eosin and Gram staining.In order to prove the establishment of PM and study the features of the disease, we performed histological analysis on the brain of moribund mice inoculated with several doses of the TIGR4 strain and sacrificed at various time-points after infection. Animals at the final stages of the disease showed typical signs of meningitis , edema, and wisps of fibrin Fig. . Inflammulp Fig. , compareulp Fig. . These dS. pneumoniae is one of the causative agents of bacterial meningitis responsible for death and sequelae worldwide. The mouse has largely proved to be a reliable animal model for studying two major pneumococcal diseases such as pneumonia [neumonia -44 and sneumonia -47. In tneumonia -9,14 andneumonia -13 have neumonia , only reneumonia ,23.Cryptococcus neoformans [S. pneumoniae, and because the use of outbred strains is cost-effective. Another research group had previously used CD-1 outbred mice in a study on the efficacy of clinafloxacin against PM; however, the authors did not provide a detailed description of the model [In the present work, starting from an experimental method used to study meningitis caused by oformans , we deveoformans  and ratsoformans , as fronoformans ,51,52 anoformans  challenghe model .S. pneumoniae strains. The strains chosen are the serotypes routinely employed by researchers in the pneumococcal field, and have proved to be highly pathogenic in different mouse infection models [50 ranging from 2.9 × 10 to 1.9 × 102 CFU. Then, PM was further characterised and standardised by using TIGR4 as a model strain. Kaplan-Meier survival analysis of animals inoculated with different doses of TIGR4 showed that 105 CFU was lethal for all mice within 48 hours from infection  in the hippocampus of a few animals.The actual induction and subsequent characterisation of PM caused by the TIGR4 strain was then carried out by histological analysis of the brain tissue from moribund animals. We chose not to analyse PM by viable counting of pneumococci in the CSF, due to the difficulty of sample collection  and to the brain . In thathe brain . In anotna magna . In thatna magna . This firocesses . In our The present work proposes a method to induce experimental PM in outbred mice by using an i.c. subarachnoidal route of infection. The stereotaxic coordinates of the injection site are provided to allow easy recognition of the inoculation point in the mouse. The model is simple and fast, and the technique assures the development of meningitis, as demonstrated by histological analysis, survival data, and microbiological parameters. No significant differences were observed in the ability of the three pneumococcal strains used to cause disease, emphasising the value of the model. It is worth noting that the use of outbred mice still results in data reproducibility, as replicates in this model closely paralleled each other in terms of survival, CFU counts per organ, and histopathological features. In addition, experiments in outbred mice are cost-effective and can be performed in larger animal groups thereby improving statistical significance. This experimental PM model may be particularly useful for all researchers involved in studies that will investigate the host-pathogen interaction at the cerebral level, with emphasis on both pathogen-associated virulence factors and host-specific brain defences.S. pneumoniae was cultured in Tryptic Soy Broth  at 37°C with 5 % CO2. Solid media were obtained by addition of 1.5 % agar and 3 % defibrinated horse blood  to TSB. When necessary, streptomycin was used at the final concentration of 500 μg/ml.Survival studies were performed with the D39, HB565 and TIGR4 strains. TIGR4 was chosen as a model strain for histological characterisation of PM and CFU counts in organs. ad libitum. All animal experiments were approved by the Local Ethical Committee . Animals were allowed to settle in the new environment for one week before performing the experiments, they were caged and given food and water S. pneumoniae strains were prepared by using a modified version of a previously described method [2O. Passaged bacteria were grown to mid-exponential phase, centrifuged for 20 minutes at 1500 × g, resuspended in fresh TSB containing 10 % glycerol, and stored in aliquots at -70°C. Numbers of bacteria were determined by viable counting of serial dilutions in sterile phosphate-buffered saline, pH = 7.4 (PBS), and plating onto blood-agar plates. Before inoculation, bacteria were thawed at room temperature, harvested by centrifugation, and resuspended in sterile PBS at the appropriate dilutions.Mouse-passaged d method . BrieflyC. neoformans in mice [PM was induced in lightly anaesthetised mice (50 mg/kg ketamine and 3 mg/kg xylazine) by modifying a method previously used to establish meningitis by  in mice ,55. Anim4 CFU per mouse were used to infect mice (n= 4) with strains D39 and HB565. In the case of TIGR4, groups of 6 to 10 animals each were inoculated with doses ranging from 10 to 105 CFU per animal. Control mice were inoculated with PBS (30 μl). Mice were closely monitored twice a day for clinical symptoms . Mice were humanely killed before reaching the moribund state. Survival was recorded for 10 days (240 hours).Different bacterial doses ranging from 10 to 10Infected mice were sacrificed either for microbiological or histological analysis. Animals were humanely killed before being moribund, and various samples were collected. For CFU counts, blood was withdrawn by cardiac puncture before sacrifice and added to a tube containing 3.8 % of sodium citrate. Brains and spleens were excised and homogenised in 2 ml of sterile PBS. Bacterial counts in blood, brain and spleen were performed by plating 10-fold dilutions onto blood-agar plates. For histopathological analysis of tissues after infection with TIGR4, brains and spleens were immediately fixed in formalin for 24 hours and then embedded in paraffin according to standard procedures. The brains were entirely sectioned along a coronal plane. Sections were stained with both haematoxilin-eosin and Gram according to standard techniques. Morphological changes were assessed by using routine light microscopy. The presence and degree of inflammation were carefully evaluated.50 values were performed by using both the method by Reed and Muench [Calculations of LDd Muench  and Probd Muench . SurvivaDC, animal experiments, microbiological analysis, writing of manuscript. ST, histological analysis, writing of manuscript. RP, animal experiments, microbiological analysis. MRO, experimental design. EB, development of methodology for induction of meningitis. MC, co-ordination of histological analysis. GP, co-ordination and design of the study, data evaluation. SR, co-ordination and design of the study, data evaluation, direct supervision of experimental work, writing of manuscript.All authors read and approved the manuscript.Document stating the ethical approval for animal experimentation conceded to the Laboratory of Molecular Microbiology and Biotechnology (LA.M.M.B.) from the University Hospital of Siena, the Medical Faculty and the Local Ethical Committee .Click here for file


Several reports indicated that non-thermal electromagnetic radiation such as from mobile phones and base stations may promote cancer. Therefore, it was investigated experimentally, whether 900 MHz electromagnetic field exposure influences lymphoma development in a mouse strain that is genetically predisposed to this disease. The AKR/J mice genome carries the AK-virus, which leads within one year to spontaneous development of thymic lymphoblastic lymphoma.320 unrestrained female mice were sham-exposed or exposed  to GSM like 900 MHz electromagnetic fields for 24 hours per day, 7 days per week, at an average whole body specific absorption rate (SAR) value of 0.4 W/kg. Animals were visually checked daily and were weighed and palpated weekly. Starting with an age of 6 months, blood samples were taken monthly from the tail. Animals with signs of disease or with an age of about 46 weeks were sacrificed and a gross necropsy was performed.Electromagnetic field exposure had a significant effect on body weight gain, with higher values in exposed than in sham-exposed animals. However, survival rate and lymphoma incidence did not differ between exposed and sham-exposed mice.These data do not support the hypothesis that exposure to 900 MHz electromagnetic fields is a significant risk factor for developing lymphoma in a genetically predisposed species, even at a relatively high exposure level. The use of mobile phones is increasing worldwide, although electromagnetic fields emitted by mobile phones and base stations are a source of great concern. However, so far it is unclear, if non-thermal exposure has a direct influence on public health. French et al.  developePim1 transgenic mice [It was suggested that non-thermal exposition to high-frequency electromagnetic fields may rather have a tumor promoter than an initiator effect , since Dnic mice , which anic mice .The differences between the studies may indicate that various species or strains as well as cancer types differ in their sensitivity to electromagnetic field exposure. The sensitivity to electromagnetic fields may result from an acquired lower resistance against adverse effects or a genetic predisposition . DiffereAKR mice are widely used in cancer research for their high leukaemia incidence (60–100%) . Mice of320 female AKR/J mice were airfreighted from the Jackson Laboratory , at an age of 4–5 weeks. After arrival, animals were randomized and housed in groups of 6 or 7 on softwood bedding  in polycarbonate cages , enriched with paper. Mice were allowed free access to mice standard food pellets  and tap-water. Twice a week cages were cleaned and water changed. Temperature was controlled at 21°C ± 2°C. The light was on a 12 hours light-12 hours dark cycle, with light on at 8 am. No sterilization measurements were taken, since AKR/J mice are not especially sensitive for pathogens. However, to prevent a possible transfection from humans to mice or mice to mice, respectively, masks and gloves were worn, which were sterilized between handling of different cages. Animals were inspected daily for signs of moribundity and were weighed (accuracy ± 0.1 g) and palpated weekly. Starting with an age of 6 months, blood samples were taken monthly from the tail. A tattoo in the ear allowed individual identification. The Bremen state commission for animal welfare according to §8a of the German animal welfare legislation approved the experiments (522-27-11/2-0).In order to accomplish whole body exposure of a large number of non-restrained animals, one approach is to design exposure setups based on radial waveguides. Inside the waveguide the animals are kept in cages which are arranged at a constant distance from a radiating antenna in the centre, thus uniform exposure of the cages can be achieved. Another advantage is that the radial waveguide is an electromagnetic shielded system, on that score no costly shielding of any laboratory is necessary. The height of the waveguide is preferably chosen smaller than half a wavelength . TherebyTherefore, special modifications of the fundamental geometry of radial waveguides had to be performed to avoid the propagation of the unwanted higher order modes. In order to produce a well defined field distribution the cage region was excited only by the fundamental TEM-mode in a first step, i.e. for small radii the plate distance was kept below 14 cm and for larger radii the height was increased to the required value of 17 cm Figure . Since iBoth implemented waveguides  of ca. 4 m diameter and 17 cm vertical plate distance were placed within the same room and carried up to 24 cages measuring 425 mm × 265 mm × 160 mm (L × W × H) each of them housing 6–7 mice. The cage area was covered with trapezoidal lids (3 cages per opening) with wire mesh . The total level was 69 dB (lin), and less than 25 dB at frequencies between 8 and 40 kHz. Thus possible disturbing effects of ultrasound were excluded.2 gas when signs of a developing disease became evident or at an age of about 46 weeks, after a last blood sample was taken. A gross necropsy was performed focusing on main tissues of disease involvement  and tumor infiltration . Tissues were immersion-fixed in Bouin's solution for up to 24 h and subsequently in ethanol (70%), embedded in paraffin and sectioned at 5 μm. Blood smears were stained with Pappenheim's stain and tissue slices using hematoxylin and eosin. When a mouse was found dead in its cage  a necropsy was performed, but no tissues were fixed.Animals were sacrificed by COGroup mean body weights were tested for a possible exposure influence in dependence of time, using multiple regression analysis . An unpaired t-test was applied to compare the loss of weight associated with lymphoma development. Survival curves and lymphoma incidence were plotted according to the method of Kaplan and Meier. Differences between curves were compared using the logrank test, with animals censored, which were still alive at the end of the study . Two-way ANOVA was used to test for possible changes in differential leucocytes counts with time and exposure.Statistical significance of differences was tested two-sided at the p ≤ 0.05 level. If not indicated otherwise, data are given as means ± standard error of the mean. The exposure code was broken only after completion of the analyses.Chronic exposure to 900 MHz electromagnetic fields had no influence on the absolute body weight of female AKR/J mice; however, its influence on the relative weight change was significant (p < 0.001) Figure . The rapWater consumption was approximately 4 g per day and mouse, and not different between exposed or sham-exposed animals (data not shown). This value is in accordance to the water intake measurements published by the Jackson Laboratory , and obvSimilar survival rates were seen in both groups of AKR/J mice Figure . The firIn female AKR/J mice lymphoma developed rapidly, usually associated with lymphadenopathy. In 28.2% (exposed) and 30.3% (sham-exposed) of all animals, lymphomas were restricted to the thymus, followed by respiratory distress and protrusion of the eyes. 13 animals  died in their cages without any earlier sign of distress, although autopsy revealed enlarged mediastinal mass compressing the thoracic space. The lung was affected in 10% of the exposed and sham-exposed animals; 8 exposed and 6 sham-exposed animals developed macroscopically visible metastatic tumors in the liver or spleen Figure . Other cWhen the animals reached an age of about half a year, differential counts of leucocytes were performed monthly using blood smears from tail blood. As seen in Figure The present study was designed to test the hypothesis that 900 MHz electromagnetic fields, modulated according to the global system for mobile communication (GSM), increases the risk of lymphoma incidence in female AKR/J mice. According to the results this hypothesis has to be rejected, since compared with sham-exposed females of this strain, a mean exposition of 0.4 W/kg SAR neither influenced the risk to develop lymphoma, nor the malignancy of the disease, only an influence on the growth pattern of the animals was observed. However, the present experiment does not allow any conclusions about tumor onset or the kinetics of tumor development, since for such type of study animals would have to be sacrificed and examined at fixed intervals irrespective of clinical symptoms.Caenorhabditis elegans [Pim1 mice was not changed due to exposure to electromagnetic fields [Exposure to electromagnetic fields increased the growth rate in the nematode  elegans , but dec elegans . In contc fields ,18. Duriad libitum, and various studies described effects of electromagnetic fields on the nervous system in humans or rodents [Food was available  rodents -39. It mMitochondrial heat production is one of the main energy consuming processes in endotherms like mice ,41. The Pim1 transgenic mice, which are known to develop spontaneous lymphoma with a high incidence rate. Lymphoma risk was found to be significantly higher in the exposed Eμ-Pim1 mice than in the controls, mostly pronounced for non-lymphoblastic lymphoma. Humans are presently not known to carry an activated Pim1 gene, but other inherited gene defects are known that predispose carriers to develop cancer [Pim1 mice from the same supplier  as Repacholi et al. [A demonstration that long-term exposure to electromagnetic fields derived from mobile phones or base stations increases the incidence of tumors in animals would provide direct evidence that such radiation is carcinogenic. The most positive evidence of an effect of exposure to high frequency electromagnetic fields similar to that used by mobile phones was reported by Repacholi et al. , using Ep cancer . Howeveri et al. , did noti et al. . Becausei et al. .A Japanese study showed that neither 1.5 GHz nor 929.3 MHz electromagnetic field exposure promotes liver carcinogenesis in a medium-term bioassay system, using partially hepatectomised F344 rats . A monopIn the present study non-restrained mice that are genetically predisposed to develop lymphoma with a high incidence were exposed to a "far-field" similar to both Australian studies ,18, withThe present study does not support the hypothesis that chronic exposure to high-frequency electromagnetic fields, similar to those emitted by mobile phones and base stations promote neoplastic development in the hematopoietic system in genetically predisposed mice. However, we cannot rule out that exposure to electromagnetic fields may be risk factors for other neoplastic development.The authors declare that they have no competing interests.AS carried out the study, performed the statistical analyses and drafted most of the manuscript. AL conceived the study, participated in its design and coordination, and drafted some parts of the manuscript. JS, AB and VH developed the technical set up and delivered the dosimetry for the experiment. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Cytology slides are often unique and irreplaceable. Unlike surgical pathology cases, where additional paraffin sections can be cut, cytology slides often cannot be duplicated because there are only a few direct smears or the diagnostic material is present on a single slide. Cytology slides are often "sent out" to other physicians, laboratories or hospitals, typically so that the pathologist at the institution where the patient will receive treatment can review the slides. Less often, a cytology lab sends out the slides for a second opinion or as part of the discovery process in a lawsuit, where they may or may not be defendants. Rarely, unique and irreplaceable cytology slides are lost. This article presents a hypothetical scenario that is based on reported state appellate court decisions. The article discusses some of the legal issues that will affect the defendant cytologist/cytology lab and the "expert cytologist," and suggests some steps a cytologist/cytology lab can take to minimize the risk of repercussions from a lost unique and irreplaceable cytology slide. A WestLaw search  uncovered only a handful of reported appellate cases that directly applied to the issue of lost cytology slides. There are many cases and statutes dealing with lost and altered evidence (other than cytology slides), but the circumstances surrounding cytology slides are unique. I did not see any previous reviews or commentary specifically addressing the topic. Notwithstanding the relative paucity of on-point legal authority, the issue is one commonly addressed by cytology laboratories and cytologists and many have approached the issue thoughtfully and prudently.This review synthesizes the available legal cases and presents to practicing cytologists a short, relatively concise summary in the format of a hypothetical case. The review seeks to incorporate the legal issues with the practical aspects of running a cytology laboratory. The review answers how some state courts might approach the problem of lost and irreplaceable cytology slides, and offers general ideas to cytologist for minimizing the risk from lost slides.The issues surrounding the decision whether to send out diagnostic patient slides are more important in cytology than in surgical pathology because the cytology slides are typically unique and irreplaceable. Unlike surgical pathology cases, where additional paraffin sections can be cut, cytology slides often cannot be duplicated because there are only a few direct smears or the diagnostic material is present on a single slide.Slides are routinely "sent out" for a variety of reasons. Most commonly, slides are sent to another institution because the patient's pathology slides will be reviewed before treatment. Some smaller laboratories with only one or two pathologist may send out slides as part of their quality assurance procedures. More rarely, but increasingly, slide's are requested as part of existing or contemplated litigation. Slides might be lost in any of these circumstances. A laboratory or hospital might loose the slides and not discover their absence until the slides are requested.This article will discuss the approach the US legal system takes in addressing what happens when cytology slides are lost, and what steps a prudent laboratory might take to manage the risk. The article also intends to improve cytopathologists' awareness and understanding of some of the legal issues that arise when evidence is missing and perhaps promote an international comparative discussion of alternative legal approaches.In the US, most of medical malpractice law is made and interpreted by state legislatures and state courts. What follows is a hypothetical story based on several legal decisions made by appellate state courts in the United States and reported in the legal literature. Although based on real cases and available to the public, the names have been changed. Importantly, none of this is intended as legal advice and readers should consult their attorney about specific questions.Rugged Labs (RL) is a small, independent laboratory. Part of Rugged Labs' work involves providing Big Giant Lab (BGL) with overflow services for cytopathology. In December of 1995, BGL bought out RL.In 1994 and 1995, a RL cytotechnician interpreted two Pap smears from 30-year-old Ms. Penny as normal. In 1996 a cervical biopsy from Penny showed adenocarcinoma. Ms. Penny underwent a radical hysterectomy, which confirmed the invasive endocervical adenocarcinoma. Ms. Penny is alive today, but endured extended post-operative hospitalization.At the time of the hysterectomy, Penny Plaintiff's oncologist requested that the 1994 and 1995 PAP smear slides be sent to Dr. Experta, who interpreted both Pap smears as containing "abnormal cell groups consistent with adenocarcinoma." Dr. Experta also reviewed the biopsy and in a note concluded that the cells on the Pap smears were consistent with the adenocarcinoma diagnosed on the cervical biopsy.Approximately 6 months later, Plaintiff Penny decided to sue for medical malpractice based on failure to diagnose her endocervical adenocarcinoma on the Pap smears.At some time before the plaintiff filed her lawsuit Dr. Experta's assistant apparently mailed the slides back to BGL. The Pap smear slides are lost, presumably in the mail, by BGL or Dr. Experta's office.RL, the independent laboratory, asked the trial court to grant it summary judgment on the basis that the evidence was lost and no questions of fact remained. Summary judgment means that there are no outstanding questions of fact and the court needs to decide only questions of law. Summary judgment means there is no trial with a jury or judge hearing and weighing evidence. The question of law RL wanted the court to determine on summary judgment was that the lost slides substantially prejudiced RL and summary judgment was, therefore, appropriate. RL included in its motion for summary judgment an affidavit from an expert stating that she could not give an opinion without having the slides to look at. An affidavit by Dr. Experta's secretary stated that the slides were returned to BGL by US mail. BGL submitted an affidavit attesting they did not lose the slides. The parties to the lawsuit stipulated that the slides were lost. The trial court agreed with RL that there should be summary judgment in RL's favor, reasoning that no questions of fact needed to be answered and that a trial would unduly prejudice RL because of the spoliation of evidence. For a summary of the facts, please see Fig. 1.The Plaintiff appealed and the state appellate court reversed the trial court, concluding that the trial court made a mistake in not allowing the case to go to trial. The appeals court reasoned that the case turned on a question of fact; "The slide either showed the presence of cancer cells or it did not." The appellate court envisioned the trial as follows: the cytotechnologist from RL would "testify to her conclusions" and the plaintiff would have Dr. Experta testify to her conclusions. There should be a trial because there were questions of fact including, in the words of the majority, whether the slide had "cancer cells" or not.The majority also discounted the affidavit from BGL, stating that the conclusory statement that BGL had not lost the slides was not valid; just because BGL couldn't find the slides did not mean they never had them. In a footnote, the majority noted that although they aren't accusing anyone, they couldn't help but notice that missing the slides benefited BGL and RL.One judge dissented in the three-judge panel that decided the case. The dissent focused on two points. First, the dissent emphasized that the defendant RL had nothing to do with loosing the slides. Although the plaintiff, Ms. Penny, had no "direct role" in the loss of evidence, the dissent treated Dr. Experta as an agent of the plaintiff and concluded that Dr Experta should have sent the slides back to RL not GBL. The dissent also reasoned that its approach would "encourage experts to treat more carefully evidence delivered into their hands."Secondly, the dissent emphasized that the defendant RL is disadvantaged by the loss of the slide and the plaintiff has gained a significant advantage. The dissent sees a trial where the cytotechnologist's faces a serious credibility problem because her testimony will come across as blatantly self-serving. The cytotech will be limited to "opining that he made no error." Moreover, the fact-finder may conclude that the cytotechnologist is wrong since many may presume that the expert pathologist must be right. Finally, the defendants cannot obtain their own expert to bolster their case, because no cytology slides remain for review.The dissent concludes that summary judgment for RL was proper because the trial court had good reasons to conclude that the lost evidence was going to unduly prejudice the defendant RL.The most obvious issue this case brings up is the difficulty in deciding what to do when it isn't clear who lost the evidence, or when a third party lost the evidence. In contrast, is the situation where one party is responsible for inadvertently losing the evidence or, even worse, where one party deliberately loses or destroys evidence. This is called spoliation of evidence and includes meaningful alteration of the evidence.The court will determine the severity of the sanction for spoliation by the degree of willfulness or bad faith and the extent of the prejudice suffered by the non-responsible party. For example, in one case, surgeons performed a hepatectomy after a small needle core liver biopsy was interpreted as cancer. The hepatectomy specimen showed only cirrhosis and no cancer was found. The small needle liver biopsy was subsequently lost by the hospital. The patient sued for medical malpractice, claiming that the core biopsy was misdiagnosed and lead to an unnecessary hepatectomy. The trial court instructed the jurors that they could "draw the strongest possible inference against [the hospital] as to what the lost cytology slides would have shown." In other words, losing the slide means your opposition can make the slide show whatever they want. Interestingly, in the hepatectomy case, the defendants prevailed by arguing that the diagnosis of cancer and the decision to undergo resection were reasonable, regardless of the cytology results, because the patient had active hepatitis B and a suspicious liver mass on imaging. They argued that even with a negative cytology result the surgeons would have gone ahead with the hepatectomy.But our case is different. Neither party, according to the majority, is responsible for losing the slides, or put another way, either party might be responsible for losing the slides. Several judicial options exist and none are neutral. One is to impose no sanctions and to proceed as usual, only without the slides, as the majority opinion advocated. This likely favors the plaintiff, particularly when there is a subsequent surgical specimen with cancer. A second option is to try to determine which party the court thinks is more prejudiced by the lost slides and then impose a legal remedy, as the trial court did in our hypothetical case by granting summary judgment. The difficulty is that it will not be clear which side is more prejudiced until the slide is recovered. The critical question of whether the negative diagnosis fell below the standard of care can likely be adequately answered only if the parties and their experts can review the slide. A third option might be to not allow Dr. Experta's testimony if the defendant can show that the expert knew or should have known that there was going to be litigation . ArguablInterestingly, each option leads to a different result based on bias about which party is ultimately more responsible or more likely responsible for loosing the slides and a bias about the standard of care. The majority's opinion included a note that the benefit to BGL from losing the slides can't be ignored, a clear statement that the plaintiff had no responsibility for losing the slides, and a simplistic view about the standard of care reflected by the statement that "the slide either showed the cancer cells or it did not." The majority's subtext is that the plaintiff did nothing wrong and they weren't completely sure about the laboratory. The dissenting opinion, in contrast, treated Dr. Experta as the plaintiff's agent and was dissatisfied that Dr. Experta returned the slide to BGL instead of RL, even though at the time BGL had purchased RL. The dissent also showed a more nuanced understanding of the standard of care, conveying skepticism about Dr. Experta's look back conclusion that the Pap smears were "consistent" with adenocarcinoma. The dissent reasoned that perhaps the cells are consistent with malignancy only in the retroscope and that a defense expert might reasonably conclude that it was "not below the standard of care to determine the biopsy negative" were the slides available for review.Penny vs. GBL, a court does not dismiss the plaintiff's case, the absence of the cytology slide will likely impact the defendant cytologist more adversely because many people will assume that the cancerous cells were on the slide and the cytologist or cytotechnologist missed the cancer, which, after all, was present on the subsequent biopsy. In the hypothetical's facts, this was particularly true since Dr. Experta had already opined that the Paps were "consistent with adenocarcinoma." The defendant cytologist/cytology lab is at a serious disadvantage if the court allows Dr. Experta's opinion as admissible evidence. The majority's comment that GBL benefited from the lost slides and the appellate court's decision to allow the plaintiff to go to trial, suggests the court's bias that they believed Dr Experta's interpretation was the correct one.The facts often surrounding a cytopathology medical malpractice case are that there is a subsequent biopsy or surgical specimen with a discrepant diagnosis. If, as in the hypothetical Admittedly, a cytology lab or cytologist does benefit if slides are lost and the court does not attach any responsibility for losing the slides to the potential laboratory or cytologist defendant, because the plaintiff will not have enough evidence to prevail. Similarly, if the slides are discarded after the legal time periods the likelihood of a successful lawsuit is slim because the plaintiff will not have enough evidence, and the defendant complied with legal requirements regarding slide retention. The hypothetical case of Penny vs GBL differs. Remember that once the court allows the case to go to trial, GBL only benefits from absent slides if the defendant initially misinterpreted the slides. If the slides contained no malignant cells, and Dr. Experta over-interpreted the slides, then GBL is prejudiced by not being able to show the slide.Cytology slides are typically in possession the cytology labs. The slides may be sent out for a variety of reasons, but at least initially the lab has possession of the cytology slides. This is both an advantage and a disadvantage for the lab. The disadvantage is that if slides are lost while in the lab's possession a court will typically see it as the lab's responsibility to safeguard the slides. This allows the plaintiff to have the jury infer whatever is best for the plaintiff's case; that the slide had malignant cells when the cytology diagnosis was benign or that there were only benign cells when the diagnosis was malignant, as in the hepatectomy case.The advantage for the cytology lab is that it is in a position of relative control. The lab can implement a system to help reduce the chances of losing a slide and reduce the risk if a slide is lost. Although the lab may want to employ the help of an attorney experienced in these matters, there are several steps every cytology lab can take. First, the lab should ensure that cytology slides are retained for the time that the current federal CLIA regulations, applicable state regulations and the CAP checklist require. All glass cytology slides must be retained for at least 5 years and fine needle aspiration slides retained for 10 years. (Some state regulations may require longer times.) The CAP checklist also requires policies for "protecting and preserving the integrity and retrieval of original slides in cytopathology" and "to ensure defined handling and documentation of the use, circulation, referral, transfer and receipt of original slides to ensure availability of materials for consultation and legal proceedings." Keeping careful records about when and which slides are released to whom is essential for reducing the risk of losing slides. In the send out cases where slides are sent out for a routine second opinion not sought in contemplation of a law suit or because a patient will be treated elsewhere, the lab might obtain the borrower's explicit written agreement that the borrower has responsibility for the slides with an explicit provision about a duty to indemnify the cytology lab for any losses due to a lost slide or slides. A documented telephone call to request the return of tardy slides may also be worthwhile. In cases where slides are requested in contemplation of a lawsuit, the lab may be able to implement a policy that review of slides is done at the lab, ensuring that the lab retains possession. Alternatively, the lab may pursue or agree to a court order requiring production of the slides that clearly addresses who is responsible for the slides and includes an indemnity clause in the event the slides are lost.It is reasonable to make a distinction between sending out non-reproducible slides to an institution that will treat the patient and sending out non-reproducible slides to the plaintiff's expert witness. It is, therefore, important that the lab understands the purpose for which the slides are requested. A documented telephone conversation may clarify the purpose of the outside review and allow the lab to appropriately "triage" the case.Laboratory administrators should understand that the cytology lab serves a public function in safeguarding cytology slides. Court's have recognized a public policy reason to have the laboratory safeguard slides, in part so that the slides are available in the event of malpractice litigation. At the same time, many states have statutes that give patients the right to examine and copy their medical records . These two propositions are not mutually exclusive. One state case considered the question of whether a patient/plaintiff had a right to "immediate possession of pathology slides" and concluded that the plaintiff did not. The court decided that the patient's rights did not exceed the patient's statutory right in the slides. In other words the court was not going to find a common law, or customary, right in the slides that gave the patient a greater right than the applicable statute. The decision noted that the legislative history of the statute included remarks that pathology slides were part of the medical record. The judges then approached the second question about what to do when the "medical record" cannot be duplicated, as with a Pap smear or other cytology slide. In answering this question, the court noted that hospitals and laboratories have public as well as private duties. One of their public duties is to retain slides so that the slides are available in the event of malpractice litigation. This meant that patients do not have a legal right to possess parts of the medical record that cannot be duplicated. The court, however, did not grant the lab complete authority to never release the slides. Since the public policy reason depended fundamentally on preserving slides to help the legal system run smoothly, the decision reminded the laboratory or hospital that, pursuant to the clear terms of a statute, it must send the original slides to a "licensed institution, laboratory or physician" at the patient's written request.Moore vs Regents of the University of California [The dissent characterized the pivotal issue differently and concluded that the patient had a right to immediate possession because the slides contained the patient's cells and she had the right "to control one's body." The dissent reasoned that recent advancement in genetic science and the accompanying difficult privacy issues raised by genetic information strengthened the patient's right to possession of the cells on the glass slide. The majority addressed this argument and, citing the well known case of lifornia , remindePenny v GBL illustrates, lost slides can result in problems for cytologists and the cytopathology laboratory A good place to start is to ensure that existing CLIA regulations, applicable state regulations and other guidelines, such as the CAP checklist, are in place in the laboratory. Thinking about the issue and implementing appropriate risk management strategies with the help of an attorney are also prudent measures.In summary, lost slides can be a problem for cytology laboratories and cytologists, whether responsible for losing the slides or not. The prudent cytologist will minimize the risk of lost slides, because, as the hypothetical case of


Binocular vision requires an exquisite matching of projections from each eye to form a cohesive representation of the visual world. Eye-specific inputs are anatomically segregated, but in register in the visual thalamus, and overlap within the binocular region of primary visual cortex. Here, we show that the transmembrane protein Ten_m3 regulates the alignment of ipsilateral and contralateral projections. It is expressed in a gradient in the developing visual pathway, which is consistently highest in regions that represent dorsal visual field. Mice that lack Ten_m3 show profound abnormalities in mapping of ipsilateral, but not contralateral, projections, and exhibit pronounced deficits when performing visually mediated behavioural tasks. It is likely that the functional deficits arise from the interocular mismatch, because they are reversed by acute monocular inactivation. We conclude that Ten_m3 plays a key regulatory role in the development of aligned binocular maps, which are required for normal vision. The visual world is represented within the brain as a series of maps of visual space. In species with binocular vision, the inputs from the two eyes are aligned to form a cohesive map; little is known about how this organisation is achieved during development. We show that a transmembrane protein, Ten_m3, plays an important role. Ten_m3 is required for the guidance of uncrossed retinal axons: uncrossed projections from the eye to the brain map aberrantly in mice that lack Ten_m3, although crossed projections map normally. Consequently, projections from the two eyes are not aligned in these mice. We show that this mismatch has devastating consequences for vision. Mice lacking Ten_m3 perform very poorly in behavioural tests of visual function. The deficits are a direct result of the mismatch, because acutely silencing inputs from one eye restores visual behaviour. This remarkable and rapid recovery suggests the mismatch of the inputs from the two eyes leads to functional suppression in the brain. We conclude that Ten_m3 acts as an eye-specific guidance cue for retinal axons and is required to produce aligned projections from the two eyes, and further, that this is critical for normal visual function. Ten_m3, a transmembrane protein, has a newly discovered role in guiding retinal axons, aligning projections from the two eyes, and thereby mediating binocular vision. The functional capabilities of the central nervous system are critically dependent on highly specific patterns of neural connectivity that are established during early development. In many parts of the brain, axonal projections form a topographic map whereby relative spatial relationships between afferent and target fields are maintained. This spatial mapping is thought to be important in maintaining the integrity of information at successive levels of processing. Molecular gradients have been shown to play a key role in establishing normal topography in the developing visual system –5. BinocDrosophila pair-rule gene Ten_m/Odz [We recently identified Ten_m3 in a microarray screen for molecules that are differentially expressed between visual versus nonvisual pathways . The Tenen_m/Odz ,26. Receen_m/Odz . Both Teen_m/Odz ,29. The p < 0.05, Pairwise fixed reallocation randomisation test [We first analysed the normal expression of Ten_m3 in the developing visual pathway using in situ hybridisation. Ten_m3 mRNA was expressed in the innermost layer of the developing retina, corresponding to the developing RGC layer at embryonic day (E)16 A. The diion test ). Withinion test  and termion test  of ipsilion test . Quantifp < 0.001, Pairwise fixed reallocation randomisation test) compared to wild type (WT), suggesting that it may be targeted for degradation rather than synthesized into protein. Western blots using an antibody directed against the extracellular domain of Ten_m3 on brain lysates from WT mice showed a single band of approximately 300 kDa corresponding to the Ten_m3 protein, whereas homozygous KO mice lacked this . Many adult Ten_m3 KO mice have a slightly curly tail and/or a humped back. The appearance of the brain is normal, and histological observations did not reveal any major changes in brain structure or organisation. Most importantly, no differences in size, cellular density, or lamination were apparent in Nissl-stained sections through retina, dLGN, or visual cortex of Ten_m3 KOs compared to the WT mice .To investigate a role for Ten_m3 in regulating the formation of visual projections, the organisation of ipsilateral and contralateral retinal projections was examined in Ten_m3 KOs during the fourth postnatal week when the projection is adult-like . RGC axon = 5; KO: 14.7% ± 1.02, n = 5; p > 0.5, t-test). It thus seems that the ipsilateral zone of the dLGN is elongated rather than expanded per se.In WT mice, the ipsilateral and contralateral projections are anatomically segregated; contralateral projections fill all regions of the dLGN not occupied by ipsilateral terminals, including the ventrolateral region of the nucleus I–3I". Gi2, n = 3; KO: 1.62 ± 0.17 mm2, n = 3; p > 0.9, t-test; n = 3) or their density within the VTC . These numbers provide an estimate of just under 1,000 ipsilaterally projecting RGCs in both WTs and KOs, similar to previously published values in pigmented mice [In mice, over 95% of RGCs project contralaterally. The adult ipsilateral projection arises from RGCs within the peripheral ventrotemporal retina known as the ventrotemporal crescent (VTC). Within the VTC, approximately 15% of RGCs project ipsilaterally and the remaining RGCs project contralaterally . In normted mice . The lacp < 0.01, Wilcoxon rank sum test, comparing ipsilateral sections in KOs versus ipsilateral sections in WT, and contralateral sections in KO and WT). The position of the contralateral patch along the DV-ML axis was similar between WTs and KOs, supporting the suggestion that there is no major shift in topography of contralateral projections. As an additional control, focal injections into the dorsal retina were also performed. These experiments revealed no change in the topography of contralateral retinal projections from this region . Follows region . No ipsi5 pixels, n = 9; KO: 6.7 ± 1.2 × 105 pixels, n = 8; p > 0.5, t-test). That is, although there may be a subtle decrease in the precision of geniculocortical connectivity, the overall topography of the pathway appeared normal in Ten_m3 KOs.The dLGN receives inputs from the retina and sends outputs to the primary visual cortex (area 17). We therefore examined the topographic relationship between visual thalamus and cortex in Ten_m3 KOs. This was of particular interest both because Ten_m3 is expressed in geniculocortical neurons and in developing visual cortex , and becIn order to confirm these results with respect to the potential transfer of information from the aberrantly mapped ipsilateral eye to the visual cortex, retrograde tracing from the cortex was combined with anterograde tracing from the retina. In WTs, injections of green CTB into the lateral (binocular) zone resulted in a patch of retrogradely labelled cells in the dorsomedial region of the dLGN. Notably, all of the retrogradely labelled cells were contained either within, or immediately adjacent to, the ipsilateral patch . To test the ability of Ten_m3 KOs to perform this task using the visual system, the task was repeated under normal (ambient) light with the whiskers trimmed so they could not rely on somatosensory cues. WTs scored significantly better than KOs under these conditions . In many cases, KOs did not reach out until their nose touched the bar .We wished to determine whether the observed retinogeniculate mismatch affects vision in the Ten_m3 KO mice. Three visually mediated behavioural tasks—vertical placement, horizontal placement, and a modified version of the visual cliff test —were per the bar B. The hon = 31), whereas Ten_m3 KO mice spent approximately half their time  in this region  and suggests that, unlike WT mice, KOs do not exhibit a preference for the patterned half of the box. A more detailed analysis of the behaviour of the mice showed that although KOs approached the cliff less often than WTs , they crossed it significantly more frequently . In addition, the average time spent on the cliff side of the box per crossing was almost 10-fold higher in KOs . As a control for possible differences in absolute activity levels, the average linear displacement of a randomly chosen subset of KOs and WTs was also determined; this did not differ between the groups of mice .The visual cliff test also revealed a clear difference in the behaviour of Ten_m3 KOs D. For ths region D. This dn = 7; KO: 925 ± 249 cm/min, n = 5, p > 0.5, t-test; time on patterned side: WT: 55.9 ± 2.9%, n = 7; KO: 54.0 ± 3.8%, n = 5; p > 0.5; approaches: WT: 17.0 ± 2.8, n = 7; KO: 17.0 ±1.4, n = 5; p = 1, t-test; percent crossings: WT: 88.7 ± 3.4%, n = 7; KO: 90.8 ± 0.9%, n = 5; p > 0.5, t-test). When these values were compared to those for each group under normal light conditions, it was found that mean activity levels were markedly higher for both groups, although this did not reach statistical significance for either WTs or KOs . The number of approaches in WTs showed no difference when compared to WTs under light conditions , but KOs approached the border significantly more often in the dark compared to light conditions . Both groups also crossed the cliff significantly more often than under normal light . Most significantly, both groups of mice spent approximately half their time on the patterned side , but essentially identical to those for KOs under ambient light. Together, these results suggest that the marked difference in the behaviour of the WTs and KOs in the visual cliff test is indeed mediated by visual cues. They also indicate that the KOs have the capacity to distinguish light from dark, even though they show little behavioural response to the visual cliff.To determine whether the observed differences in the behaviour of WTs and KOs in the visual cliff test are mediated by visual cues, the same test was conducted under dark conditions (red light). The two groups performed almost identically to each other for all parameters examined , suggesting that the drug did not compromise the mobility of the mice. The number of approaches to the cliff made by WT mice with monocular TTX (n = 6) showed no difference to WT mice without TTX . The proportion of times they crossed the border  and total time spent over the patterned surface  were, however, both noticeably different. Although the differences were not statistically significant, the change in behaviour associated with monocular TTX was of concern.We postulated that, given the relatively normal histological appearance of the visual pathway, the defect in the performance of the visual cliff test may be a direct result of the interocular mismatch in the retinogeniculocortical pathway see  rather tn = 6), were associated with the mouse crossing the border when the cliff was predominantly within the field of view of the active eye. Expanding the criteria to include cases where the cliff was within the field of view of both of the active and inactive eye produced similar results . These values are not different from the percent crosses made under normal light conditions . In contrast, a very high proportion of approaches made where the active eye was facing away from the cliff were associated with a crossing . This value is significantly higher than the percent crosses under normal light , and the percent crosses made when approaches were made using the active eye  or both eyes , and is in fact similar to the percent crosses made under dark conditions. These results show that the decrease in the performance of the WTs with monocular TTX is directly associated with the loss of part of the visual field, rather than with other effects of the drug. The time spent on the cliff side of the box per cross did not show a significant difference compared to untreated WTs .To see whether the decrease in the performance levels of the WTs with TTX was a direct consequence of the loss of part of the visual field, the data were analysed to see whether the behaviour of the mice correlated with the eye that was facing the cliff as they approached the border. For example, the mice often approached the cliff either at an acute angle, or close to one of the side walls. If the active eye was facing away from the cliff, or towards the wall, the cliff would be largely or completely out of the field of view of the active eye. The number of approaches, and associated crosses, were therefore subdivided into those made while the cliff was predominantly within the field of view of the active versus the inactive eye. When the analysis criteria were restricted such that only approaches where a clear bias for one eye versus the other was apparent, we found that only a minority of these  to KOs without TTX  and appeared to actively investigate the cliff before retreating (n = 7). This value is significantly less than for KOs without TTX . The KOs with TTX also exhibited a more than 10-fold reduction in the time spent over the cliff per crossing compared to KOs without TTX , a value similar to WTs under normal light. On average, KOs spent 90.6 ± 7.9% (n = 6) of time on the patterned side during monocular blockade, almost identical to the values obtained for WTs under normal conditions. The difference between this value and that obtained from the KO mice under control conditions is significant . As with the WTs, the KO mice crossed the cliff much less frequently when it was in the field of view of the active versus the inactive eye . To confirm that this remarkable apparent recovery of visually mediated behaviour was due to the monocular blockade rather than individual variation, two KO mice were tested both before and during the blockade. Both of these mice spent far less time on the cliff side  during the monocular blockade than before the blockade.The behaviour of the Ten_m3 KO mice following monocular activity blockade was dramatically altered in comparison to their behaviour without TTX. Under these conditions, the KOs approached the border a similar number of times  The visual cliff test employs the ventral visual field , which is not part of the binocular visual field in mice . (2) TheBased on our anatomical data, we suggest that the mistargeting of ipsilateral axons in the dLGN in Ten_m3 KOs results in a more widespread representation of ipsilateral inputs to the visual cortex rather than being confined to the lateral third of area 17 as in WTs . Cells tAll experiments were performed on mice. Protocols were approved by the animal ethics committees of University of Sydney, Australia, Massachusetts Institute of Technology, United States of America, and/or Lund University, Sweden, and conformed to the National Institutes of Health and the National Health and Medical Research Council guidelines.http://www.perkinelmer.com). Antibody staining was performed using a rabbit anti-Ten_m3 [http://www.vectorlabs.com) and a TSA plus (fluoroscein) kit.Embryos were obtained from timed matings of C57/Black6 mice. Mothers were anaesthetised with 4% isofluorane and the embryos delivered by Caesarean section and then decapitated. Neonatal mice were anaesthetised with an overdose of sodium pentobarbital and the brains removed. For PCR analysis, the retinas were subdivided and relevant regions were dissected. RNA was extracted, and quantitative real-time PCR for Ten_m3 and a control gene was performed as described in . For in i-Ten_m3  at 1:25,−/−Ten_m3 mice were generated with a targeting construct in which the transmembrane-containing exon 4 of the Ten_m3 gene was disrupted with a neomycin gene (+/−Ten-m3 and subsequently homozygous Ten_m3−/− (KO) mice. Third generation backcrosses into C57/Bl6 were performed, but KO mice did not survive well on this background. Consequently, these mice were crossed with Sv129 mice, and the strain was maintained on this background. Animals are of varied pigment. Because alterations in the ipsilateral pathway of pigmented and albino mice have been reported [cin gene . The tarreported , all quareported .All surgical manipulations were performed under anaesthesia induced and maintained by inhalation of 2%–4% isofluorane in oxygen. Intraocular injections of 0.5–1 μl of 1% CTB conjugated to Alexa Fluor 594 (red) or Alexa Fluor 488 (green) were made into the right and left eyes of Ten_m3 KO and WT littermates at P21–23. For combined anterograde and retrograde labelling, injections of green CTB were made into subregions of visual cortex and red CTB was injected into one eye as above. Animals were euthanised 1–3 d later and perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Coronal sections, 60 μm thick, were cut using a freezing microtome. Focal injections of a 10% solution of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) in dimethyl formamide were made in peripheral VT retina of P11–12 mice under isofluorane anaesthesia; animals were perfused 1–2 d later. This age was chosen because topography is largely adult-like by this stage . CoronalImages were analysed using Image J (NIH). The freehand selection tool was used to outline the perimeter of the dLGN, and the area contained within this region in arbitrary units was determined. Analyses were performed across the entire rostrocaudal extent of the nucleus. Care was taken to exclude the optic tract, intergeniculate leaflet, and vLGN from this analysis. To isolate ipsilateral projections, background was subtracted using the rolling ball function, and the area containing the ipsilateral projections was selected using the wand tool. To determine shifts along the DM-VL axis, the image was cropped at the borders of the dLGN, thresholded as above, and the coordinates of each non-zero pixel were written to a file along with information on the size of the image in arbitrary units. The DM-VL axis was divided into 100 bins, and the proportion of label in each bin, expressed as a percentage of the total ipsilateral label contained within each bin, was plotted using Matlab. Means and standard errors for five KOs and five WTs were calculated and plotted. For the analysis of focal injections, all sections containing label from four KO and four WT animals were used. Thresholded data were binned as above, and Matlab was used to generate a heat map of the distribution of label. For an objective analysis of patch number, the Image analysis toolbox (Matlab) was used to calculate the number of clusters of label in images from KO and WT animals from the thresholded images. The area of labelled regions in BDA-injected animals was calculated by thresholding all labelled images in ImageJ. Labelled pixels were written to a text file and summed across all labelled sections for each animal. Means and standard errors are presented unless otherwise stated. For determination of the area of retina containing a high proportion of ipsilaterally projecting RGCs, this region was outlined in low-power photomicrographs of retinal whole mounts using ImageJ. For estimates of cell density, two fields of view within the region containing ipsilaterally projecting RGCs were photographed using the 20× objective for three KO and three WT cases. Images were thresholded to isolate individual RGCs. The number of thresholded patches was calculated using ImageJ software.−2) light with whiskers trimmed and under red light with whiskers at normal length. For the horizontal placement test, mice were moved horizontally toward the bar and scored as above.For the vertical placement test, mice were held by the tail and lowered towards a horizontal metal bar. Scores were given by two independent observers who were blind to genotype, according to when mice made a clear reaching motion for the bar with their forepaws. A score of 2 was given for mice that reached more than 1 cm from the bar and a score of 1 for mice that reached within 1 cm, but before their nose touched the bar. A score of 0 was given for mice that touched the bar with the nose before reaching for the bar. The vertical placement test was performed under 2 conditions: normal  base. A high-contrast grating was attached to the underside of one half of the box. The box placed such that the clear half of the base protruded from the laboratory bench, revealing a drop to the floor of approximately 90 cm. Mice were placed in the centre of the box and their behaviour monitored for 10 min via a digital video camera mounted 1 m above the floor of the box. The data were analysed, blind to genotype, from video recordings. An approach was defined as moving from the patterned region towards the “cliff” such that the nose was within 5 cm of the midline. Crossing was defined as the animal completely crossing the midline from the patterned to the clear half of the box. Retreating was defined as moving such that the head was within 5 cm of the midline and retreating without the entire body crossing the border. Subsequent approaches were not scored until the mouse had moved outside the 5-cm range. To confirm the role of the visual system in this task, the test was also performed under dark (red light) conditions. The effect of monocular blockade of activity was determined by injecting 0.5 μl of 1 mM tetrodotoxin into the left eye under isofluorane anaesthesia as above. Some mice appeared lethargic for a few hours postinjection and so were allowed to recover from any systemic effects of the procedure for 2–16 h before testing to ensure that this would not bias the results. Activity levels were monitored to confirm this was the case by calculating the movement of the mice for 1 min. For this, a scaled grid was placed over the video monitor, and the points at which the mouse crossed the grid were plotted and measured. In addition to the other analyses performed, the approaches and crosses during monocular inactivation were subdivided into cases where the mouse approached the cliff such that it was predominantly in the visual field of the active versus the inactive eye. Approaches to the cliff that were made such that the mouse was parallel to and within 10 cm of a side wall, and thus the cliff was largely out of the field of view of the eye facing towards the wall, were included in this analysis. Approaches made at an angle such that the cliff was predominantly within the field of view of one eye rather than the other were also included. Approaches where there was no clear bias for one eye versus the other were scored separately. The effectiveness of the blockade at the time of the test was confirmed by checking that the pupillary light reflex was absent in the injected eye.Figure S1Sections through the dLGN following a focal injection of DiI into the dorsal retina in a WT (A) and a KO (B) mouse. Terminals are seen in ventral dLGN in both cases. Scale bar indicates 100 μm.(1.3 MB TIF)Click here for additional data file.Figure S2p > 0.5, t-test; n = 9 for WT and n = 8 for KO). Scale bar in (D) indicates 200 μm, which applies to all images.Coronal sections through the dLGN following injections of the neuronal tracer BDA into medial (A) and (B) or lateral (C) and (D) area 17 in WTs (A) and (C) and KOs (B) and (D). A dense patch of label containing retrogradely labelled cells and anterogradely labelled corticothalamic fibres (arrows) is seen in the dLGN (borders indicated by dashed line). The positions of the labelled regions are similar following injections into a particular part of the visual cortex: medial injections label ventral dLGN (A) and (B), whereas lateral injections label dorsal dLGN (C) and (D). The labelled area appears slightly larger in Ten_m3 KOs, but this difference was not significant ((5.1 MB PDF)Click here for additional data file.Video S1The mouse is allowed to move freely around a box with a clear acrylic base. A contrast grating is appended below half of the box. The animal pauses when it reaches the border between the patterned and nonpatterned halves, appears to inspect the border region but does not cross over, then retreats to the patterned region.(1.2 MB MPG)Click here for additional data file.Video S2Unlike the WT mouse, when the KO mouse approaches the border region, it crosses onto the nonpatterned side with no hesitation.(1.1 MB MPG)Click here for additional data file.Video S3Under these conditions, the KO mouse appears to inspect the border region and retreats to the patterned side. This behaviour is very similar to that observed in WT mice under normal conditions see .(7.7 MB AVI)Click here for additional data file.http://www.ncbi.nlm.nih.gov) accession number for Ten_m3 is NM_011857.The National Center for Biotechnology Information (NCBI) (


Survival remains an issue in pulmonary hypertension, a chronic disorder that often affects aged human adults. In young adult mice and rats, chronic 50% hypoxia (11% FIO2 or 0.5 atm) induces pulmonary hypertension without threatening life. In this framework, oral dehydroepiandrosterone was recently shown to prevent and reverse pulmonary hypertension in rats within a few weeks. To evaluate dehydroepiandrosterone therapy more globally, in the long term and in old age, we investigated whether hypoxia decreases lifespan and whether dehydroepiandrosterone improves survival under hypoxia.240 C57BL/6 mice were treated, from the age of 21 months until death, by normobaric hypoxia (11% FIO2) or normoxia, both with and without dehydroepiandrosterone sulfate (25 mg/kg in drinking water) . Survival, pulmonary artery and heart remodeling, weight and blood patterns were assessed.In normoxia, control mice reached the median age of 27 months . Hypoxia not only induced cardiopulmonary remodeling and polycythemia in old animals but also induced severe weight loss, trembling behavior and high mortality . Under hypoxia however, dehydroepiandrosterone not only significantly reduced cardiopulmonary remodeling but also remarkably extended survival . Weight loss and trembling behavior at least partially remained, and polycythemia completely, the latter possibly favorably participating in blood oxygenation. Interestingly, at the dose used, dehydroepiandrosterone sulfate was detrimental to long-term survival in normoxia .Dehydroepiandrosterone globally reduced what may be called an age-related frailty induced by hypoxic pulmonary hypertension. This interestingly recalls an inverse correlation found in the prospective PAQUID epidemiological study, between dehydroepiandrosterone blood levels and mortality in aged human smokers and former smokers. In human beings, pulmonary hypertension (PH) is a chronic and life threatening disorder in which a progressive increase of pulmonary vascular resistance leads to right ventricular failure. When detected, PH is often an already irreversible chronic pathology and leads to death after several years of severe illness and treatment -5. AmongTherapies under development may be studied in rats and mice by their effects on pulmonary arterial pressure or cardiopulmonary remodeling. Survival has been studied in rats with the use of monocrotaline injection to model PH -17, but Therefore we decided to use hypoxia, up to death in mice, starting at an age when they naturally start dying, in order to evaluate long-term positive or negative survival effects of hypoxic PH and a potential therapy. We considered dehydroepiandrosterone (DHEA), that has recently been shown to prevent and treat chronic hypoxic PH in rats when administered orally in its free  or sulfHypoxic pulmonary vasoconstriction helps oxygenating the blood but increases pulmonary arterial pressure. By relaxing contracted pulmonary arteries ,26,27, DMice were obtained at the age of 17 months  and randomly distributed into 4 groups (N = 60) in cages containing 7 to 9 mice each with ad libitum standard diet  and water. At the age of 21 months – which we will refer to as t = 0 – each group received a different environmental condition, defined by a combination of hypoxia or normoxia and DHEA or not. Cages were changed weekly and food and drink renewed every other week. All procedures concerning animal care and use were carried out in accordance with the European Community Council Directive (86/609/EEC). All animal procedures were approved by the animal care and use committee at the institute. All treatments and measures were performed by investigators blinded to the treatment.We chose normobaric hypoxia (11% FIO2) to avoid potential harmful consequences of rapid pressure variations. Hypoxic mice were housed in a home-made chamber homogeneously supplied by a flow of a filtered mixture of air and nitrogen  at ambient pressure and 11 ± 1% oxygen . Control normoxic mice were housed in a similar chamber supplied by a flow of filtered air. Gas flowed sufficiently fast (15 l/min) into the chambers to ensure low carbonic gas levels (less than 0.05%). Hypoxia was interrupted weekly for roughly one hour for animal care.DHEAS  was incorporated at 0.25 mg/ml  into thSurvival was checked every one to three days until t = 180 days . From time to time mice were weighed and their food and drink consumption was approximated by giving 350 g food and 500 ml drink per cage and measuring how much remained one week later.Cardiopulmonary remodeling was measured in mice that died before t = 90 days . Right ventricular hypertrophy was assessed by the right ventricle to left ventricle plus septum weight ratio (RV/LV+S) . Lungs wBlood sampling was performed on one initially randomly chosen cage per group. Additional cages were randomly chosen if needed to have at least 5 mice tested per group. The mice to be tested were placed in clean cages with their usual drink but no food overnight, and were excluded from survival analysis. Blood sampling (300 μl) was performed retro-orbitally under inhaled isoflurane anesthesia, in the morning. Blood was mixed with 10% ethylenediaminetetraacetic acid at 0.5 M. A blood analyzer  provided hematocrit, hemoglobin content, and the count, volume and hemoglobin concentration of red blood cells.Values are expressed as mean ± SEM. Statistics were performed with JMP 6.0 . Comparisons between two and several groups were done by Student and one-way ANOVA tests, respectively. Survival curve characteristics and comparisons were based on the proportional hazards Cox model. The method for choosing the number of animals is provided in an online additional file see .Survival is clearly the main global health indicator. Note that mortality may affect the significance of results by death selection.It is rather unusual to start lifespan experiments with animals that are already aged. We wanted to start treatment (hypoxia or normoxia and DHEA or not) when the rate of 'natural death' becomes signifiant in C57BL/6 laboratory male mice. Starting with mice that are too old would imply that selection by death has comenced and that only resistant mice are being studied. If the mice are too young then in the short term no natural death will occur and any survival improvement due to a therapy may not be detected.It appeared from the literature ,32 that Survival curves are shown in Figure Over the next 3 months of treatment . Pulmonary artery remodeling  is shown in figure Compared to the control group, hypoxic mice had higher pulmonary artery  and heart  remodeling. DHEA had no effect on the normoxic cardiopulmonary system but under hypoxia DHEA significantly reduced pulmonary artery and heart remodeling .Overall, the mean daily consumption was of 3.0 ± 1 g and 3.25 ± 0.28 ml per mouse, with no particular distinction over groups and time. The consumption may have been lower because the determination did not take into account food and drink remaining at the bottom of the cage, which depend on the number of mice per cage, on their activity and on cage manipulation during the week. For DHEA-treated mice weighing ~30 g, we estimate that the DHEAS consumption was on the order of 25 mg/kg/day.Sick mice generally lose weight and as such body weight figure  may be uWhen the mice arrived, we observed that they were thin . The mice regained normal appearance within a month. When measured two months before treatment, all groups had similar weights (27.9 ± 0.12 g) and food and drink consumption.Weights of control mice gradually increased until the age of 25 months . After two or three months, the -remaining- mice regained normal size  at t = 5 weeks and t = 5 months. The same trend was observed for red blood cell counts and blood hemoglobin content, while cellular hemoglobin content remained unchanged.DHEAS did not affect the hematocrit nor red blood cell properties, neither in normoxia nor in hypoxia, at t = 5 weeks and t = 5 months.1. Hypoxia induced PH in old mice and DHEA prevented it. 2. Hypoxia drastically induced mortality and weight loss in old age. 3. In its sulfate form and at the used oral dose DHEA was detrimental to long-term survival in normoxia. 4. DHEA however largely prevented hypoxic death during the whole experiment.This is not particularly surprising as it also does it in young adult mice  and ratsDHEA has already been shown to prevent and reverse PH in rats ,24. DHEAPH in old age. Old age is a common factor for PH incidence and low endogenous blood DHEA(S) levels in humans ..We are nAlthough DHEAS administration appeared to be detrimental in the long term , and although hypoxic animals treated by DHEA still lost weight and trembled, DHEA largely (but not completely) prevented the hypoxic mortality over the whole experiment. This overall beneficial survival effect is the best possible answer to our questions: DHEA not only treats hypoxic PH but also hypoxic (old) mice.The vasorelaxation of pulmonary arteries by DHEA could have led to overall negative effects since hypoxic vasoconstriction of pulmonary arteries is useful to improve blood oxygenation. The question arises of whether, with DHEA treatment, the body managed without the oxygen provided by vasoconstriction or another mechanism for providing an adequate oxygen supply came into play. The high blood hemoglobin content here may play a role. By preventing cardiopulmonary remodeling but permitting increased hematocrit under hypoxia, DHEA could be favorable to the animal's health by preventing heart failure (due to PH) while allowing high oxygenation.The prevention of hypoxic death by DHEA in mice recalls us the prospective PAQUID study in humans, where a strong inverse correlation between natural DHEA(S) blood levels and the ten year mortality in old male smokers and former smokers has been reported [reported . There ireported . TherefoThere seems to be a frailty to hypoxic PH that is particular to old age, in mice and possibly in humans. This suggests that survival studies with aged mice under hypoxia may be pertinent for evaluating therapies for aged patients having PH. In that framework, DHEA was found to remarkably improve survival under hypoxia. The comparison between mice and humans is not obvious, but our findings interestingly resemble human observations, that together suggest trials of DHEA treatment to PH and COPD in humans.FIO2: Fraction of Inspired OxygenPH: Pulmonary HypertensionCOPD: Chronic Obstructive Pulmonary DiseaseDHEA(S): DeHydroEpiAndrosterone (sulfate)This work was financed by the Association pour la Recherche sur les Nicotianés .EHD carried out the design of the study, performed the statistical analysis, carried out the environmental setting, participated in blood analysis, anatomopathological analysis and drafted the manuscript. JQ carried out the anatomopathological analysis and helped to design the study. EEB participated in design and coordination of the study and helped to draft the manuscript.SurvivalPowerClick here for file


Alzheimer's disease (AD) is a progressive neurodegenerative disorder that primarily strikes the elderly. Studies in both humans and animal models have linked the consumption of cholesterol and saturated fats with amyloid-β (Aβ) deposition and development of AD. Yet, these studies did not examine high fat diets in combination with reduced carbohydrate intake. Here we tested the effect of a high saturated fat/low carbohydrate diet on a transgenic mouse model of AD.Starting at three months of age, two groups of female transgenic mice carrying the "London" APP mutation (APP/V717I) were fed either, a standard diet (SD) composed of high carbohydrate/low fat chow, or a ketogenic diet (KD) composed of very low carbohydrate/high saturated fat chow for 43 days. Animals fed the KD exhibited greatly elevated serum ketone body levels, as measured by β-hydroxybutyrate (3.85 ± 2.6 mM), compared to SD fed animals (0.29 ± 0.06 mM). In addition, animals fed the KD lost body weight . In contrast to earlier studies, the brief KD feeding regime significantly reduced total brain Aβ levels by approximately 25%. Despite changes in ketone levels, body weight, and Aβ levels, the KD diet did not alter behavioral measures.Previous studies have suggested that diets rich in cholesterol and saturated fats increased the deposition of Aβ and the risk of developing AD. Here we demonstrate that a diet rich in saturated fats and low in carbohydrates can actually reduce levels of Aβ. Therefore, dietary strategies aimed at reducing Aβ levels should take into account interactions of dietary components and the metabolic outcomes, in particular, levels of carbohydrates, total calories, and presence of ketone bodies should be considered. Alzheimer's disease (AD) is an age-associated neurodegenerative disease that is very common in the US, affecting up to 50% of people between the ages of 75 to 84 years . The numThe development of AD and the accumulation of Aβ have been linked to dietary factors. Diets rich in saturated fat have been repeatedly implicated in epidemiological studies -8, thougad lib and variable in composition. Despite these differences, low carbohydrate diets may also be effective in preventing seizures and may work through similar mechanisms as a KD  C57Bl × FVB female mice of 3 months of age were used for this study. Mice were housed under a reversed day-night rhythm: 14 hours light/10 hours darkness starting at 7 p.m. in standard metal cages type RVS T2 (area of 540 cm2). The number of mice per cage was limited in accordance with legislation on animal welfare. All mice were genotyped by polymerase chain reaction (PCR) at the age of 3 weeks. Mice were blind randomized and age-matched and had free access to pre-filtered and sterile water (UV-lamp). Mice had free access to either ketogenic (KD)  or standard (SD) chow . The F3666 chow is a runny paste and was given in special designed liquid food suppliers and was refreshed daily. F3666 is a liquid chow and the animals frequently spilled the chow in the cage. Also, since all the animals in a given group were housed together in a single cage, measuring chow intake per animal was not possible and was not recorded. Due to some problems with weight loss in animals in the KD group, these animals were fed a mixed chow 1(SD):3(KD) starting at day 16 until day 20. From days 21–27 the amount of SD chow was reduced to a few crumbs sprinkled over the KD chow. After day 28 the animals were returned to KD chow only. However, one mouse in the KD group refused food intake and died despite attempts of feeding via gavage. One control animal died during blood draw.Blood was collected from anesthetized mice from either the orbital plexus or via a heart puncture. β-hydroxybutyrate levels were measured spectrophotometrically using the Stanbio liquicolor β-hydroxybutyrate kit .et al. [The novel object recognition test was performed after 38 days of treatment using the method described by Dewachter et al. . Briefly® (Ketamin), Rompun® (Xylazin 2%) and Atropine (2:1:1) and flushed trans-cardially with physiological serum at 4°C. This was performed to remove blood from the brain vessels, a procedure which has no influence on organ integrity. Blood was collected via a heart puncture and a 1 ml syringe in heparinized Eppendorf tubes. The brain was removed from the cranium and hindbrain and forebrain were separated with a cut in the coronal/frontal plane. The forebrain was divided evenly into left and right hemisphere by using a midline sagittal cut. One hemisphere of each animal was homogenized using a Potter, a glass tube  and a mechanical homogenizer (650 rpm). A volume of 6.5 × 1/2 brain weight of freshly prepared 20 mM Tris/HCl buffer (pH 8.5) with Proteinase Inhibitors  was used as homogenization buffer. The homogenates were collected in Beckman centrifuge tubes TLX and collected on ice prior to centrifugation. Before the samples were centrifuged, 10 % of the sample was used for determination of the total protein concentration of the homogenate whereas 90 % of the samples were centrifuged to process further for biochemistry. The supernatant (soluble fraction containing secreted APP and amyloid peptides) was separated from the pellet by centrifugation (membrane fraction containing membrane-bound APP-fragments). The supernatant was processed further by column chromatography to concentrate the amyloid peptides using small reversed phase columns . Amyloid peptides were eluted with 75% A-TFA and the eluates were collected in 2 ml tubes on ice. Eluates were freeze-dried in a speedvac concentrator  overnight and resolved in 240 μl of the sample diluent furnished with the ELISA kits. To quantify the amount of human Aβ40 and human Aβ42 in the soluble fraction of the brain homogenates, commercially available Enzyme-Linked-Immunosorbent-Assay (ELISA) kits were used . Quantification of the Aβ content of the samples was obtained by comparing absorbance to a standard curve made with synthetic Aβ40 or Aβ42. Total protein concentration in the brain homogenate was measured using Bradford solution (Pierce Inc.).The mice were anaesthetized with a mixture of KetalarAD, Alzheimer's disease; APP, amyloid precursor protein; Aβ, amyloid beta; SD, standard diet; KD, ketogenic diet; CR, caloric restriction; BHB, β-hydroxybutyrate.As co-founder of Accera, Inc., STH holds shares in an organization and may gain or lose financially from the publication of this manuscript. In addition, STH has applied for patents relating to the content of the manuscript and may gain or lose financially from publication of this manuscript.


Declining reproductive performance is a serious breeding concern in many countries. To reveal the situation in Norwegian cattle, trends in reproductive performance were studied using insemination reports from 1985 to 2005 and data based on herd recording files from 1989 to 2005. The total number of first services was 469.765 in 1985 declining to 335.712 in 2005. The number of recorded herds and animals declined from 21.588 to 14.718 and 360.289 to 309.452 from 1989 to 2005, respectively. Sixty days non-return rate after single inseminations (NR60) increased from 68.1 in 1985 to 72.7% in 2005 (p < 0.001) and the number of services per inseminated animal (NIA) decreased from 1.8 to 1.6 (p < 0.001) from 1985 to 2005. However, return rates 0–3 days post insemination (RR0-3) increased from 6 to 12% in the same period (p < 0.001). NR60 was higher and the RR0-3 was lower in the summer season compared to the winter season during the whole period. A fertility index (FS), has been calculated from the herd recording files each year from 1989 to 2005. The average FS-index did not show a significant trend and the calving interval was also fairly constant between 12.4 and 12.6 months during this period. The average interval from calving to first and last insemination, respectively, increased from a low of 79 and 102 days in 1990 to a high of 86 and 108 days in 2005. Both intervals were consistently longer for cows in first lactation than for cows in later lactations. The percentage of inseminated animals reported culled because of poor fertility decreased from 6.0% in 1989 to 4.6% in 1996 and thereafter again increased to 6% in 2005. In conclusion, most fertility measures, mainly comprising the Norwegian Red (NRF) breed, show a relatively high level of reproductive performance with a positive or a relatively constant trend during the last two decades. In many countries there has been a decline in reproductive performance in dairy cattle. Several studies show increasing number of days from calving to first service and decreasing pregnancy rates, e.g. -5. As a The results obtained in the present study are based on insemination reports and herd recording files in Norway comprising 66.8% of the herds in 1985 increasing to 94.2% in 2005  declininTrends concerning age of heifers at first insemination, average number of days from calving to first (CFI) and last insemination (CLI), respectively, number of animals inseminated (I), calving interval and animals culled because of failure to breed (AC) were obtained from herd recording files from 1989 to 2005. During this period there was a decline in number of recorded herds from 21.588 to 14.718 and animals from 360.289 to 309.452. A fertility index, Fertility status (FS), was also calculated for each herd from the herd recording files every year from 1989 to 2005. FS index is expressed by the formula:Comparisons of NR60, RR0-3 and NIA between years or groups were performed using chi-square analysis.th year from 1985 to 2005 is shown in figure st services remained similar  and cows with >1 lactation (n = 127252) were 1.5, 1.8 and 1.7  respectively for controlled animals in 2005. Data from 1989 to 2005 show each year similar differences in NIA between heifers, cows in 1st and >1 lactation. Fig Figure The percentage of inseminated animals reported culled because of poor fertility is shown in Fig et al. [et al., studying phenotypic and genotypic trends in heifers and first lactation cows [st lactation and >1 lactation cows were 68.8, 56.0 and 58.7% respectively. These results show that the pregnancy rates in NRF is relatively high when compared to studies from many other countries, e.g. [The aim of the present study was to describe some trends in reproductive measures in Norwegian cattle the last two decades. Since NRF has been by far the most dominant breed during this period, the data presented mainly reflects the reproductive performance of this breed. To describe the fertility trends, 60 days non return rates and number of services per inseminated animal are used among others. As a measure of fertility, non return rates have some disadvantages, as described by Salisbury et al. . Cows, oion cows . Howeverion cows ,5. The nion cows . In thises, e.g. -17.The improvement in NR60 is probably caused by a variety of reasons, one of them being the breeding strategy, which gives increasing weight to fertility and health traits. In Norway, fertility has been emphasised in the total merit index from the 1970's based on progeny testing utilising large daughter groups of the NRF breed . Other rThe increasing trend in CFI, and consequently also CLI, is probably mainly caused by managerial factors and farmer decisions. However, partly it is probably also caused by a small and undesirable genetic change for CFI, which has been observed in first lactation cows . In thisst lactation animals, but also >1 lactation cows, is in accordance with the differences in pregnancy rates after single first inseminations registered by Refsdal et al. [The study shows that reproductive performance in Norway is consequently higher in the summer months compared to the winter season. This is in contrast to many countries under subtropical and tropical conditions experiencing decreased fertility in dairy cows inseminated during the hot summer months -29. The l et al. . The FS-l et al. ,30.In conclusion, most fertility measures in Norwegian cattle, mainly comprising the NRF breed, show a relatively high level of reproductive performance and a positive  or relatively constant trend  during the last two decades. This is probably caused by a variety of reasons, one of them being the breeding strategy, which gives increasing weight to fertility and health traits. However, the interval from calving to first and last insemination, respectively, has slightly increased during the period and the RR0-3 has increased. The calving interval has been relatively constant in spite of increasing non return rates and lower number of services per animal inseminated, mainly because of a longer interval from calving to first insemination. This is also the main reason why the FS-index has been relatively constant. In contrast to many countries under subtropical and tropical conditions the reproductive performance in Norway is higher in the summer months compared to the winter season. This pattern has been similar over time and is probably caused by a variety of environmental factors.