Identifying marathon runners at risk of neurological deterioration at the end of the race  is challenging. Here we report a case of rehydration-related hyponatraemia with ensuing brain herniation.We report the death of runner in his 30's who collapsed in the recovery area following a marathon. Following rehydration he developed a respiratory arrest in the emergency room. He was found to be hyponatraemic (130 mM). A CT brain scan showed severe hydrocephalus and brain stem herniation. Despite emergency insertion of an extraventricular drain, he was tested for brainstem death the following morning. Funduscopy demonstrated an acute-on-chronic papilledema; CSF spectrophotometry did not reveal any trace of oxyhemoglobin or bilirubin, but ferritin levels were considerably raised , consistent with a previous bleed. Retrospectively it emerged that the patient had suffered from a thunderclap headache some months earlier. Subsequently he developed morning headaches and nausea. This suggests that he may have suffered from a subarachnoid haemorrhage complicated by secondary hydrocephalus. This would explain why in this case the relatively mild rehydration-related hyponatremia may have caused brain swelling sufficient for herniation.Given the frequency of hyponatraemia in marathon runners (serum Na <135 mM in about 13%), and the non-specific symptoms, we discuss how a simple screening test such as funduscopy may help to identify those who require urgent neuroimaging. Rehydration-related hyponatraemia and immediate death from brainstem herniation after a marathon is exceedingly rare . In contA male aged in his 30's was admitted to the Emergency Room following collapse in the recovery area following a marathon. He had completed the marathon within around 4 hours on a sunny but cold day. In the Emergency Room he felt faint and dizzy and complained of a headache. His GCS was 15/15, pupils were reactive and his general medical examination was normal. He had a blood pressure of 130/70 mmHg, the ECG showed sinus rhythm of 80 bpm, his chest X-ray did not show any evidence for pulmonary oedema. His blood glucose was 5.4 mM and the Na was 133 mM. He was one of hundreds of athletes presenting at the same time with very similar symptoms, thought to be related to dehydration, and consequently received intravenous rehydration (1 L of 5% Dextrose and 1 L 0.9% NaCl). Four hours after his initial collapse he suddenly vomited and his GCS dropped to 11/15. Shortly after this he suffered a respiratory arrest requiring tracheal intubation. An urgent CT scan showed midbrain herniation into the foramen magnum Figure  and 1F aA CSF sample was taken during the operative procedure and sent for spectrophotometric assessment of pigments to evaluate whether this could be a CT-negative SAH with secondary hydrocephalus. CSF spectrophotometry did not reveal any trace of oxyhaemoglobin or bilirubin , but ferThis tragic case illustrates several problems which are frequently seen in the Emergency room: hyponatraemia, headaches and nausea.Rehydration-related hyponatraemia occurs in about 13% of athletes  making iWe therefore suspect that our patient may have already suffered from a SAH, producing substantial hydrocephalus prior to the race, thus allowing for only for a small degree of parenchymal brain swelling leading to herniation. It has been suggested that hyponatraemic encephalopathy be named Varon-Ayus syndrome .Any newly developing severe headache is suspicious. A SAH should be suspected if the headaches are of sudden onset and associated with vomiting, meningism (neck stiffness) and a rise in blood pressure. All of this was noted in our case. Characteristically 50% of patients having a SAH experience an instantaneous, thunderclap headache and about 20% will recall similar headaches in the preceding days . MeningiRaised intracranial pressure (ICP) should be suspected if nausea (morning sickness) is associated with headaches, loss of appetite and chronic optic disc oedema. The development of optic disc oedema can be classified into four stages: early, developed, chronic and atrophic . Early oProbably the most reliable clinical signs of optic disc oedema are swelling of the NFL, peripapillary retinal NFL hemorrhages and blurring of the peripapillary NFL Figure . The sweBecause of individual differences, retinal hyperemia and blurring of the disc margins are less reliable clinical signs and it is most unlikely that one will have seen (and remember) a particular disc of a patient who presented to the Emergency Room in the past. Typically, acute optic disc oedema arises bilaterally and is associated with full visual fields, normal visual acuity and color vision. The only finding may be an enlarged blind spot .In contrast to acute optic disc oedema, absent spontaneous venous pulsation (SVP) is an unreliable sign for high ICP, because SVP is only observed in 80% of normal subjects .3, no CSF white cells were seen). This rendered the CSF:serum albumin ratio as an indicator for integrity of the blood-CSF barrier questionable. Therefore we did not measured the CSF albumin in this case. The high CSF lactate of 12.7 mM  together with the normal CSF total protein and glucose  suggested an increased anaerobic metabolism, possibly due to high ICP and poor CNS perfusion.In this case the CSF analysis provided important diagnostic clues. Firstly spectrophotometry did not show any trace of haemoglobin, oxyheamoglobin or bilirubin. This virtually excluded a recent bleed . The norThe key result in this case was the 44-fold elevated CSF ferritin of 530 ng/mL. It has previously been shown that CSF ferritin, which is too large to pass through the blood-CSF barrier (450–480 kDa), is produced intrathecally ,19. CSF In summary, a targeted CSF analysis consisting of at least cytology, CSF total protein, glucose, lactate, CSF spectrophotometry and CSF ferritin levels can provide important clues for the diagnostic work up of patients presenting to the Emergency Room with suspected CNS pathology causing non-specific symptoms such as headaches and nausea.In conclusion, a simple clinical test such as funduscopy in the Emergency Room may allow for early identification of those athletes who require neuroimaging. With the benefit of hindsight it may have been possible to have suspected a SAH or the presence of intracranial hypertension on the basis of the clinical signs and symptoms in this case.CT = computer tomorgraphy; CSF = Cerebrospinal fluid; GCS = Glasgow Coma Scale; ICP = intracranial pressure; NFL = nerve fiber layer; SAH = subarachnoid haemorrhage; SVP = spontaneous venous pulsation.The author(s) declare that they have no competing interests.AP obtained consent for publication, examined the patient, performed the CSF analysis and reviewed the literature, obtained permission for publishing the personal communications and wrote the manuscript. GK contributed to the data analysis and edited the manuscript. IA examined the patient, contributed to data analysis and co-wrote the manuscript. All authors read and approved the final manuscript.Written, informed consent for publication was obtained from the next of kin. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


There was history of episodic pulsatile tinnitus for the last 1 year. There was no history of headache, vomiting, visual symptom, or head injury. Neurologic examination was normal. An ophthalmologic assessment revealed normal fundus, visual acuity, and visual field (by perimetry). Magnetic resonance imaging (MRI) of brain demonstrated a leak in the cribriform plate into the anterior ethmoid cells [2O and a normal cell count, protein, and glucose, confirming the radiological diagnosis. The patient was treated with acetazolamide and advised surgical closure but the patient denied.A 35-year-old obese woman with body mass index of 33 kg/mid cells . In addiid cells , elongatid cells  were alsIIH presenting as CSF rhinorrhea is very rare. There isMRI finding in IIH include flattening of the posterior sclera, an empty sella, distension of the perioptic subarachnoid space, enhancement of the prelaminar optic nerve, vertical tortuosity of the orbital optic nerve, and intraocular protrusion of the prelaminar optic nerve. The longIn our case, MRI of the optic nerves and pituitary fossa provided important clues to the diagnosis of IIH. Our patient showed distension of the perioptic subarachnoid space, vertical tortuosity of the orbital optic nerve, and complete empty sella on MRI, from which a diagnosis of IIH was strongly suspected and CSF pressure measurement was therefore performed. To the best of our knowledge, there have been no reports of idiopathic intracranial hypertension presenting as CSF rhinorrhea without any other classical symptoms of IIH.CSF opening pressure is not always measured for those with spontaneous CSF rhinorrhea, when the results of initial neuroimaging are grossly normal. Therefore, attention to the optic nerves and pituitary fossa should be given to the MRI of patients with spontaneous CSF rhinorrhea. In all suspected cases, CSF pressure should be measured and treatments of the IIH are warranted before the surgical repair of CSF leak.


Previously we found that the Ishikawa endometrial cancer cell line expresses macrophage colony-stimulating factor (M-CSF) and c-fms transcripts and that its proliferation is enhanced by the addition of recombinant M-CSF. This suggested that Ishikawa cells are constitutively stimulated by M-CSF. In support of this we now show that Ishikawa cells secrete M-CSF and that known stimulators of M-CSF production increase the amount detected in Ishikawa cell conditioned medium. Using retroviral infections to introduce and express exogenous c-fms genes in Ishikawa cells we also demonstrate proliferation to be partially inhibited by a dominant negative, mutant c-fms gene, yet enhanced approximately 3-fold by a normal c-fms gene, under conditions in which the only source of M-CSF was that produced by the cells. The data provide evidence for the existence of an active M-CSF/receptor loop in these endometrial cancer cells and suggests the possibility of such activity in tumours of the endometrium and ovary that aberrantly express M-CSF and fms genes.


Reports from Thailand suggest that a growing number of people who inject drugs (IDU) are now injecting midazolam, a legal benzodiazepine with potent amnestic and ventilatory depressant effects. We therefore sought to examine midazolam injection among a community-recruited sample of Thai IDU.We examined the prevalence and correlates of midazolam injection among 252 IDU participating in the Mitsampan Community Research Project, Bangkok, using multivariate logistic regression. We also examined the use of midazolam in combination with other drugs.252 IDU participated in this study, including 66 (26.2%) women. In total, 170 (67.5%) participants reported ever having injected midazolam, and 144 (57.1%) reported daily midazolam injection in the past six months. In multivariate analyses, a history of midazolam injection was independently associated with using drugs in combination , younger age , having a history of methadone treatment , and binge drug use . The drugs most commonly used in combination with midazolam were heroin (72.3%) and yaba (methamphetamine) (30.5%).We observed a high rate of midazolam injection among Thai IDU. Midazolam injection was strongly associated with polysubstance use and binge drug use, and was most commonly used in combination with both opiates and methamphetamines. Our findings suggest that midazolam injection has become increasingly common within Thailand. Evidence-based approaches for reducing harms associated with midazolam injection are needed. Studi effects -10. Mida effects . However effects . A study effects . This tr effects .It has been suggested that midazolam injection, partly because of the associated amnestic effects, can result in elevated rates of risk behaviour, including syringe sharing . The injThe Mitsampan Community Research Project is a collaborative research project involving the British Columbia Centre for Excellence in HIV/AIDS , the Mitsampan Harm Reduction Center , the Thai AIDS Treatment Action Group , and Chulalongkorn University . During July-August 2008, the partners undertook a cross-sectional study involving 252 community-recruited IDU. The primary aims of the study were to assess drug use and HIV risk behaviors and to assess barriers to access to healthcare among local IDU. Potential participants were recruited through peer-based outreach efforts and word of mouth. Study participants were invited to attend the Mitsampan Harm Reduction Center (MSHRC) to participate in the study. The Mitsampan Center was established in the Mitsampan neighborhood, which is home to large number of illicit drug users and low-income residents. Individuals were eligible for participation in this study and defined as an "IDU" if they reported injection of illicit drugs in the past six months. All participants provided informed consent and completed an interviewer-administered questionnaire eliciting information about demographic characteristics, drug use, HIV risk behaviour, criminal justice system exposure, and experiences with health care. All participants were given 250 Baht (approximately $7 USD) upon completion of the questionnaire. The study has been approved by the Research Ethics Boards of the University of British Columbia and Chulalongkorn University.2 test. We then examined factors independently associated with a history of midazolam injection use by fitting a multivariate logistic regression model that included all variables that were associated with midazolam injection at the p ≤ 0.05 level in univariate analyses. All p-values were two-sided. We also asked participants who reported midazolam injection about the frequency of their midazolam injecting in the previous six months, and the drugs they used (if any) in combination with midazolam.Using univariate statistics and multivariate logistic regression, we compared IDU who did and did not report a history of midazolam injection. Variables considered included: median age (< 36.5 years or ≥ 36.5 years), gender, education level (< secondary school vs. ≥ secondary school), heroin injection (yes vs. no), yaba  injection (yes vs. no), use of drugs in combination (yes vs. no), syringe borrowing (yes vs. no), syringe lending (yes vs. no), non-fatal overdose (yes vs. no), binge drug use (yes vs. no), having had drugs planted on oneself by police (yes vs. no), incarceration (yes vs. no), compulsory treatment experience (yes vs. no), and methadone treatment (yes vs. no). Use of drugs in combination refers to use of more than one drug at the same time . We considered experiences of drug planting by police given that this type of contact with police could potentially prompt some IDU to obtain midazolam, given that the drug can be obtained "over-the-counter" in selected pharmacies and acquiring it may involve little or no contact with the illicit drug market. This variable was ascertained by asking participants "Have police ever planted drugs on you?" Binge drug use refers to periods when drugs are used more often than usual. All behavioural variables refer to lifetime history . To examine the bivariate associations, we used the Pearson χIn total, 252 IDU participated in this study, including 66 (26.2%) females. The median age of participants was 36.5 years. Two hundred and thirty-eight (94.4%) participants were born in the Bangkok Metropolitan Area. In total, 170 (67.5%) participants reported that they had injected midazolam previously and, of these, 144  reported daily midazolam injection in the past six months. As indicated in Table In the present study, we found that approximately 68% of a community-recruited sample of IDU in Bangkok had injected midazolam previously. Fifty-seven percent of the sample had injected midazolam at least once a day in the past six months. Midazolam injectors were more likely to report using drugs in combination, binge drug use, and a history of methadone treatment. Midazolam injectors tended to be older, and were less likely to be female. Sixty-five percent of midazolam injectors reported use of drugs in combination, with heroin and yaba being the drugs most commonly used with midazolam.The prevalence of midazalom injection found in the present study is much higher than most previously reported rates ,12. Van Midazolam injection was strongly associated with the use of drugs in combination and was reportedly most commonly used in combination with both heroin and methamphetamine. This raises concern regarding the potential elevated risk for overdose as a result of polysubstance use ; howeverThe findings of this study have implications for harm reduction practice. First, because midazolam filtrate is highly acidic and damaging to veins, midazolam injectors are known to resort to groin injection when peripheral veins are no longer usable. Groin injection carries significant risk, including risk for deep vein thrombosis, pulmonary embolus, abscesses, and puncture of the femoral artery, vein, or nerve . TherefoThis study has limitations. Previous studies have indicated that the majority of midazolam is distributed in Bangkok , and theIn summary, we found extremely high rates of midazolam injection among a cohort of Thai IDU in Bangkok. Midazolam injection was strongly associated with the use of various drugs in combination and binge drug use. Given the many adverse effects of midazolam injection, evidence-based pubic health interventions are urgently needed to reduce the harms associated with this form of drug use.The authors declare that they have no competing interests.TK, NKA, NF, KH, PS, KK and EW designed the study. CL conducted the statistical analyses. TK drafted the manuscript and incorporated all suggestions from co-authors. All authors made significant contributions to the conception of the analyses, interpretation of the data, and drafting of the manuscript. All authors have read and approved the final manuscript.


Leukocytosis and thrombocytopenia occur during cardiopulmonary bypass (CPB) with extracorporeal circulation (ECC). Elevated circulating concentrations of macrophage colony-stimulating factor (M-CSF) are reported during thrombocytopenia and leukopenia of different origins. We have assessed M-CSF concentrations in 40 patients undergoing CPB with ECC. Plasma M-CSF concentrations were stable during ECC and increased at the 6th (7.3 ± 0.7 IU/μg protein) and 24th (8.6 ± 0.8 IU/μg protein) postoperative hour compared with pre-ECC values (4.9 ± 0.5 IU/μg protein). A deep thrombocytopenia was found during ECC and until the 24th postoperative hour. A drop of leukocyte counts was found during ECC followed by an increase after ECC weaning. While no correlation was found between M-CSF concentrations and the leukocyte counts, M-CSF values were positively correlated with platelet counts only before and during ECC. Thus, M-CSF is not implicated in the thrombocytopenia and the leukopenia generated during CPB with ECC. However the elevated levels of M-CSFa few hours after the end of ECC might play a role in the inflammatory process often observed after CPB.


Pleurodeles waltl has been studied extensively as a model for sex determination and differentiation. However, the evolution of its testis after metamorphosis is poorly documented.In numerous Caudata, the testis is known to differentiate new lobes at adulthood, leading to a multiple testis. The Iberian ribbed newt Pleurodeles waltl of different ages reared in our laboratory. Testis evolution was studied by several approaches: morphology, histology, immunohistochemistry and RT-PCR. Surgery was also employed to study testis regeneration.Testes were obtained from Pleurodeles waltl possess multiple testes made up of two lobes. The second lobe appears at the caudal extremity of the first one from residual primordial germ cells located near or even inside efferent ducts in the glandular tissue that usually appears following spermatozoa extrusion. Surprisingly, in the case of surgical elimination of the anterior part of the testis, de novo spermatogenesis is stopped in the first lobe which becomes restricted to the glandular tissue. Following first testis lobe removal, the anterior part of the testis regenerates a new testis lobe, a process stimulated in the presence of DHT.In this species, the testis is linked to the lung. This association consists of connective tissue derived from the mesorchium and the coelomic epithelium surrounding the lung and takes place at the end of larval life. This tissue contains lobules including primordial germ cells with a typical large and polylobular nucleus. The anterior part of the testis remains thin and undifferentiated while the posterior part differentiates in a large first testis lobe where spermatogenesis occurs during the first year of life. The undifferentiated status of the anterior part is attested by the lack of expression of the testis marker Dmrt1 and the meiosis entry marker Dmc1. Three-year-old Pleurodeles waltl constitute an original gonochoristic vertebrate model in which testis differentiation is observed up to adulthood. Like in most vertebrate species, ovarian or testis differentiation in amphibians takes place from an undifferentiated bipotential organ. Two areas can be observed in the undifferentiated gonad . At the Salamandra salamandra [Salamandra, one testis can contain up to 6 lobes.Among amphibians, most Caudata are atypical regarding sex differentiation because males possess two multiple testes: at least this is known in Salamandridae and Plethodontidae, two families representing 80% of Salamandroidea -18. Thislamandra . In thislamandra . The glalamandra . In old Pleurodeles waltl, the Iberian ribbed newt found in Portugal, Spain and Northwest Africa, is a member of the Salamandridae family. Larvae possess gills and live underwater until they metamorphose into air-breathing forms possessing lungs. Sex is determined genetically  and gonad development takes place mainly during larval life  was prepared at 3% in absolute ethanol (w/v) and stored at 4°C. Animals were anaesthetized by immersion in water containing 1% (v/v) of this stock solution.For operative procedures (either section between the posterior testis lobe and the undifferentiated anterior part or ablation of the first testis lobe), a 1.5 cm incision was made in the ventrolateral side of the abdomen and the testis was manipulated using sterile fine forceps and scissors. Animals were allowed to recover on moist paper for 24 hours. Then, they were placed in tanks containing 2 l of water supplemented or not with dihydrotestosterone (DHT). A stock solution of DHT was prepared in absolute ethanol (5 mg/ml) and stored at room temperature. For treatment, DHT was used at 400 μg/l whereas control males were exposed to ethanol. The water (with or without treatment) was renewed after each feeding.Several months after surgery, animals were anaesthetized, sacrificed, gonads and lungs were subjected to a morphological examination before histology or molecular biology studies.Tissues were fixed in Carnoy's solution, embedded in paraffin and sectioned at 7 μm. For histological studies, sections were stained with hematoxylin-eosin-light green. Proliferating Cell Nuclear Antigen (PCNA) detection was performed using the PC10 monoclonal antibody and the LSAB2 kit  according to manufacturer's instructions.For BromodeoxyUridine (BrdU) incorporation, BrdU (Sigma-Aldrich) was diluted at 1 mg/ml in Steinberg's solution and 1 ml was used for intraperitoneal injection per juvenile male. After 24 hours, the animals were anaesthetized with benzocaine and testes were harvested and fixed in Carnoy's solution. Once embedded in paraffin and sectioned at 7 μm, BrdU detection was performed using a monoclonal antibody  according to manufacturer's instructions and the LSAB2 kit.2). When circularity index = 1, nuclei are perfectly round. Cross section parameters were area and circularity index.Preparations were analysed under white illumination on a Eclipse 80i microscope . Images were collected using LuciaG software 4.81 (Laboratory imaging) which was also used to measure morphometric parameters on germ cells as well as testis cross sections. Nuclear parameters were the perimeter and the circularity index (4πarea/perimeterThe testis was separated into three parts: anterior (part linked to the lung), medium (between this anterior part and the first lobe) and posterior (the first differentiated lobe). Total RNA was extracted from each part using 200 μl of TRIzol reagent  and quantified. The detailed protocol for reverse transcription has been described previously . BrieflyTaq DNA Polymerase (Invitrogen) in PCR buffer containing 25 mM of each deoxynucleotide triphosphate, 1.5 mM MgCl2 and 10 pmol of each primer in a total volume of 25 μL. Specific primers and conditions of amplification for GAPDH , VASA, DMRT1 (doublesex- and mab-3-related transcription factor 1) and DMC1 (disrupted meiotic cDNA 1) cDNAs have been described previously [The amplification was performed with 0.5 U eviously ,25. PCR We observed that in males, each testis was linked to the lung of the same body side Figure. . This liAt the end of larval life , germ cells leave the cortex and invade the medulla all along the cephalo-caudal axis of the gonad which is a long and thin band of tissue. At this early step of gonadal differentiation, testis and ovary can be distinguished only by histological techniques: germ cells are maintained in the cortex of the ovary but they localize in the testis medulla .At the end of metamorphosis , a small enlargement appears at the posterior region of the organ: it constitutes the first testis lobe. The longest part of the testis is made up of a long and thin band of tissue that links the first testis lobe to the lung.Between metamorphosis (6 months) and sexual maturity (1.5 – 2 years), the first testis lobe grows while the length of the anterior part of the testis linked to the lung reduces  with a round nucleus containing condensed chromatin. The cortex was greatly reduced in this region and the somatic cells with small nuclei were absent. The perimeter of the largest region of the first lobe was 10.37 ± 0.17 mm as determined by the analysis of cross sections stained for histology. The first testis lobe was not circular (circularity index = 0.76 ± 0.01), due to the presence of the mesorchium and efferent ducts located in a dorsal hilum. It contained lobules showing an increasing maturity throughout the cephalo-caudal axis: the cephalic part contained proliferating spermatogonia Figure.  while moSeveral molecular markers were studied by RT-PCR in a 9.5-month-old male Figure. . We alsoIn two-year-old males, the anterior part of the testis that links the first lobe to the lung was more and more reduced Figure. . HoweverThese germ cells seemed to be quiescent up to the third year. At this moment, they become spermatogonia which together with somatic cells will generate a second testis lobe at the caudal extremity of the testis and it appears translucent Figure. . The lobversus 17 ± 0.8 mm for the contralateral testis n = 2 males): it contained mainly the glandular tissue corresponding to lobules after spermatozoa extrusion and there was no further spermatogenesis  whereas the first differentiated lobe of the testis was maintained. The experiment was performed on one testis while the other one was preserved. The analyses performed on two males 11 and 13 months respectively after surgery, showed that the operated testis was dramatically reduced in size , eight months (n = 3) or 15 months (n = 2) after the surgery, a new testis lobe was found near the lung on the side where the anterior part of the testis had been maintained Figure.  whereas Then we tested if androgens could be involved in the regulation of testis regeneration. Adult males aged between 2 and 3.5-year-old were subjected to bilateral elimination of all the testes except the anterior region linked to the lung. At the time of surgery, 2-year-old males possessed a first lobe with an anterior part containing early spermatogenic stages, a medial part with spermatozoa and a glandular part whereas 3.5-year-old males possessed the first lobe and the beginning of a second one developing caudally  are associated with the lungs. Lungs are paired ventral derivatives of the foregut and their development depends on interactive signalling between the endodermal epithelium and mesenchyme derived from splanchnic mesoderm [Pleurodeles waltl although the presence of primordial germ cells at the anterior part of the testis is well known [Ambystoma mexicanum (Ambystomatidae) and Ichthyosaura (= Mesotriton = Triturus) alpestris . In neotenic larvae of Ambystoma mexicanum, the lungs display more compartments than those of Pleurodeles waltl whereas Ichthyosaura alpestris possesses smooth-surfaced lungs [Pleurodeles waltl. We found only one sentence in a short report about Triturus cristatus: "In case of bilateral castration a few islands of germ cells intimately linked to the lung or to the kidney cannot be extirpated" [In mesoderm -28. Gonall known ,30. Thisll known . Studiesed lungs ,32. Our irpated" . This suPleurodeles waltl is regionalized. Although the migration of the germ cells from the cortex towards the medulla occurs all along the cephalo-caudal axis of the testis, in juvenile animals, the differentiation will appear first in the posterior region that evolves in a first testis lobe where spermatogenesis will take place. Our results show clearly that in the anterior part of the testis, germ cells have the morphological features of primordial germ cells (large polylobular nucleus) rather than those of spermatogonia (round nucleus). This is consistent with previous reports on Pleurodeles waltl indicating the presence of primordial germ cells at the anterior part of the testis [Cynops pyrrhogaster, Dmc1 expression was found to be absent from spermatogonia whereas the maximal expression was observed in primary spermatocytes at the leptotene-zygotene stage [The second interesting finding reported in our study is the fact that testis differentiation in e testis . After ae testis ,34,35. Se testis . Neverthe testis . In the ne stage . Despitene stage . The numne stage . AlthougWe have shown that the first testis lobe is reduced to glandular tissue when the first differentiated lobe is separated from the anterior part of the testis. This demonstrates clearly that the undifferentiated part of the testis near the lung is a source of germ cells which enter progressively into the first testis lobe together with somatic cells to constitute lobules and cysts. This is in accordance with the well known increasing maturity of the lobules throughout the cephalo-caudal axis of the testis . This isCynops pyrrhogaster and Triturus cristatus and described as testis regeneration [Triturus cristatus, regenerates were studied up to 21 months after the bilateral castration and several oocytes could be found in the gonad [The high potency of the anterior part of the testis linked to the lung to differentiate new testicular structures has been demonstrated in our study by the elimination of the first differentiated testis lobe, triggering the formation of a new testis lobe in which a complete spermatogenesis can occur. A similar result was obtained in neration ,33. In tin vivo changes in steroid synthesis could be associated not only to testis regeneration but also to lifelong testis differentiation. However, little is known about androgen synthesis in Pleurodeles waltl. A measure of sex steroid levels in the plasma by radio-immunoassay showed two annual peaks in october-november and in march with testosterone being the major steroid in addition to DHT and androstenedione [Cynops pyrrhogaster [The regenerating process appeared slower in older males (2- to 3.5-year-old instead of 16-month-old). However, this slower process allowed us to show very clearly a stimulation by DHT when added to the rearing water. This suggests that enedione . Howeverenedione . Togetheenedione , this reenedione . Of courhogaster ,43.Pleurodeles waltl, males possess multiple testes. Such an organ is a common feature of most Caudata belonging to the Salamandroidea order. Among the Salamandridae family, it was described for instance in various genus of the Pleurodelinae subfamily  [Triturus marmoratus [Notophthalmus (= Diemyctylus) viridescens, Taricha torosa (= Diemyctylus torosus = Triturus torosus) [Cynops pyrrhogaster [Euproctus platycephalus (= rusconi), Tylotriton verrucosus and Calotriton (= Euproctus) asper [Salamandra salamandra (= maculata) [Desmognathus fuscus [D. monticola, D. ochrophaeus and D. quadramaculatus [Eurycea bislineata [Chiropterotriton mosaueri [Ichthyophis tricolor and Uraeotyphlus cf. narayani [Our study also shows that in almatus) ,6, Triturmoratus , Notophttorosus) ,12, Cynohogaster , Euprocts) asper  as well aculata) ,14. Mults fuscus ,13, D. maculatus ) and theslineata , Chiroptmosaueri ). A siminarayani .Pleurodeles waltl, the way of formation of the new testis lobe which appears at the posterior extremity of a previous one, seems to be identical to what has been described in Salamandra salamandra [Desmognathus fusca [Pleurodeles waltl, we found primordial germ cells in the connective tissue surrounding efferent ducts and sometimes in the lumen of those ducts. This observation confirms previous reports [Pleurodeles waltl testis containing cysts with spermatozoa, we observed rarely primordial germ cells. The hypothesis of a migration along the efferent ducts from the anterior part of the testis is interesting but we could not test it. However, germ cells could also have another origin. Indeed, from experiments using estradiol benzoate treatment of adult males of Cynops pyrrhogaster, it has been proposed that germ cells could differentiate from the columnar epithelium surrounding the slender cords which connect testis lobes [In lamandra  or in Deus fusca . The ger reports ,47. Howeis lobes .Pleurodeles waltl. It has been proposed that in the early glandular tissue, after spermiation, Sertoli cells degenerate and they could differentiate into duct cells. This is suggested by the presence of a common basal lamina [We have no information about the origin of somatic cells in the new testis lobe of l lamina  and the l lamina . The facl lamina ,47.Besides, the factors that trigger the formation of a new lobe are unknown although androgens seem to be good candidates as suggested from our castration experiments.Pleurodeles waltl constitutes an original gonochoristic vertebrate model in which testis differentiation is observed up to adulthood. Indeed, the differentiation of gonads at adulthood occurs mainly in hermaphroditic teleost fish which are either protandrous  or protogynous (e.g. honeycomb grouper) [Pleurodeles waltl might be used for the study of factors required to maintain primordial germ cells in adults as well as factors triggering their differentiation. This could shed light on the highly debated recent finding of germline stem cells in the mammalian ovary [Like other Caudata, grouper) ,52. Pleuan ovary .The authors declare that they have no competing interests.SF designed the study, performed surgical experiments, organ collection, analyzed histological sections and drafted the manuscript. HD realized molecular biology analyses. DC performed steroid treatments and contributed to histological analysis. AC obtained data about molecular tools and critically reviewed the manuscript. All authors read and approved the final manuscript.


The worse results were observed in the composite resin group (58.05% median). Fracture strength was higher in feldspathic porcelain (196N median) when compared to resin composite (114.9 N median). All the fixed prostheses made of fiber-reinforced composite resin detached from the abutment teeth before fracturing, suggesting that the adhesive surface's retainer should be increased. The purpose of this paper was to investigate, by means of marginal adaptation and fracture strength, three different types of single retainer posterior fixed partial dentures (FPDs) for the replacement of a missing premolar. Two-unit cantilever FPDs were fabricated from composite resin, feldspathic porcelain, and fiber-reinforced composite resin. After luting procedures and margin polishing, all specimens were subjected to a Scanning Electron Microscopic marginal evaluation both prior to and after thermomechanical loading with a custom made chewing simulator comprising both thermal and mechanical loads. The results indicated that the highest score of marginal adaptation, that is, the closest score to 100% of continuous margins, at the  Two-unit cantilevered fixed partial dentures (FPDs) may be defined as retainers holding one or more unsupported free-end extensions. This type of prosthodontic rehabilitation has been used as an interim solution for restoring edentulous areas prior to and during implant therapy, instead of using a removable prosthesis . While mAmong the tooth-colored restorative materials available, fiber-reinforced composite resin is increasingly being used in prosthodontic rehabilitation –13. The The major advantages of two-unit cantilever-inlay FPDs for single tooth replacement are that they involve less tissue damage, they are easier to clean, they are less explensive and that there is no chance of undetected debonding due to its single retainer . In addiA high success rate of clinical longevity can be expected from two-unit cantilevered resin-bonded FPDs made of nickel chrome alloy . MeanwhiThe materials used in the present study are listed in n = 6). The apex of each root was sealed with an adhesive system  without removing the pulpal tissue. To simulate intratubular fluid flow, a cylindrical cavity was drilled 1.5 mm below the cementoenamel junction until the pulp chamber was reached. A metal tube with a diameter of 1.4 mm was luted into the cavity with the same adhesive system. Subsequently, the teeth were mounted on aluminium bases with micro hybrid composite-resin and the bases were immersed in an autopolymerizing acrylic resin  to an apical depth of two thirds of the root length to create a strong load-resistant support. Through a connecting silicone tube, the pulp chamber was evacuated with a vacuum pump  and then filled with a bubble-free mixture of horse serum  and phosphate-buffered saline solution  with the aid of a 3-way valve, and finally connected to a serum infusion bottle. This bottle was placed vertically 34 cm above the specimen to simulate the normal hydrostatic pressure of 25 mm Hg within the tooth. Caries-free human molars of nearly identical size and complete root growth were were procured from a private dental office with the understanding and oral consent of the patient. The teeth needed to be extracted due to periodontal reasons. They were stored in a 0.1% thymol solution for a maximum of 2 months after extraction. The teeth were randomly divided into three groups  mounted on a red contra-angle handpiece  under continuous water-cooling. The depth of the occlusal inlay was of 2 mm and the occlusal step was 4 mm. The interproximal step of 2 mm was used along with an axial depth of 1.6 mm and a faciolingual width of 3.5 mm  was made in each molar by the use of a rotating diamond instrument  cantilever FPDs, polyether impressions  were made using a simultaneous mixing technique following the manufacturer's instructions. Then, provisional restorations were made using Fermit N (IvoclarVivadent) without any cement and placed according to the clinical recommendations proposed by the manufacturer. The FRC system consists of two materials: glass fibers with different orientation  and a microfilled composite  for the veneering of the framework. The design of the fiberglass framework was first premodelled with a light polymerizing resin  to obtain the oval shape and its thickness checked on the molding model. This model was embedded in a transparent silicone impression paste to form a mold. The resin was removed, and the fibers were applied into the silicone-mould. The pre-impregnated “pontic” fibers were condensed in a deep-drawing polymerization process. After a cycle of vacuum-forming and after polymerizing by light in a special unit  for 10 minutes according to the manufacturer's recommendations, the FRC was airborne particle abraded  using a small grain size of 27 mm at 2.5 bar of pressure for 10 seconds and treated with a silane coupling agent . A sheet of wave fibers “frame” was placed upon the “pontic” structure and the cycle in the light curing unit (VS1) was repeated. The Adoro material was built incrementally and precured. The final polymerization/tempering were performed by means of light and heat . The additional tempering step at 104°C was done to maximize the strength and the surface quality of the restorations. For the construction of the feldspathic porcelain (FP) and composite-resin (CR) fixed prostheses an optical impression was made with the digital camera from the Cerec System . The construction and milling of the prostheses was carried out using the Cerec 3 system (Software version 1.60 R980) according to a modified version of a protocol for the fabrication of three-unit Cerec prostheses. Feldspathic porcelain  and microhybrid composite  prefabricated blocks were the materials used for the construction of the prostheses. After the milling procedure, they were manually adjusted to the abutment tooth using coarse diamond instruments under continuous water cooling. μm) at a pressure of 2 bars. The intaglio surfaces of the FRC and CR abutments were also abraded following the precedent procedure but for 10 seconds. The intaglio surface of the ceramic group (FP) was etched with 5% hydrofluoric acid  for 60 seconds followed by the application of a silanecoupling agent . The tooth surface, that is, enamel margin and airborne particle abraded adhesive-covered dentin, was treated using an adhesive system  after phosphoric acid selective enamel conditioning. A microhybrid light cured composite resin  was used as the luting agent  for 60 s each from cervical, buccal, lingual, and occlusal surfaces. The margins of the restorations were then finished using 15 mm diamond instruments  and polished with flexible discs . In the case of the FRC prostheses, the provisional restorations made of Fermit were removed and the teeth's dentin surfaces  were airborne particle abraded  for 2 seconds using aluminum oxide powder (grain size of 27 ng agent . An ultrAfter polishing of the margins (before loading) and after loading, the specimens were cleaned with rotating nylon brushes  and toothpaste before making impressions for the replicas. One pair of replicas from both interproximal and occlusal boxes  was proctotal margin length were analyzed on the SEM: approximal enamel, cervical enamel, and occlusal enamel  and gold sputtered  for their observation in a Scanning Electron Microscope . A quantitative evaluation of the marginal adaptation was performed at a 200x magnification by using a custom made module programmed within image processing software . Three margin segments that constituted the l enamel . The perThe specimens were mechanically loaded in a computer-controlled masticator with 1,200,000 cycles of 49N each, at a frequency of 1.7 Hz. A total of 3,000 thermal cycles of type 5°C to 50°C to 5°C were performed simultaneously. The chamber was automatically emptied after 2 minutes for 10 s with air pressure to avoid mixing the cold and warm water. The load cycles were transferred to the buccal cusp of the pontic. After the thermomechanical loading procedure, the fracture strength of each prosthesis was calculated by loading them to failure in a universal testing machine . The force was applied on the center of the pontics using a steel ball (5 mm diameter) at a crosshead speed of 1 mm/minute. To ensure a regular force distribution and minimize the transmission of local force peaks from the steel bar to the pontics' cusps, a layer of 0.5 mm thick tin foil was placed between both surfaces. The failure determination was set at a 10% loss of the maximum loading force. Radiologic examinations  were made to document the different fracture patterns. Types of failure due to the fracture strength test were described as “adhesive” (in the adhesive interface) and “cohesive” . Two main locations of the “adhesive” failures were distinguished: between luting composite and inlays' restorative material  and between tooth substrate and luting composite.The evaluation of the data was performed with Stata 9.0 for Windows. Shapiro-Wilk W test showed that the distribution of the data was not normal. Therefore, a Kruskal-Wallis test was used to detect whether there were differences in the median values of marginal adaptation at both TC and CI interfaces. Chi Square test was used to detect differences in fracture strength among groups feldspathic porcelain (VM) and composite resin (CR). Fiber reinforced composite group (FRC) was excluded from statistical analysis as will be explained in the results section. Multiple comparisons between groups were carried out with Bonferroni post hoc test.tooth-composite interface, no significant differences were detected among FP, FRC, and RC prior to loading. The percentages of continuous margins were of 97.25, 97.65 and of 95.75, respectively. After loading, FP showed significantly better results (88.1% of continuous margins) when compared with CR (58.05% of continuous margins), as detailed in The results of marginal adaptation and fracture strength  are detailed in Figures composite-inlay interface were above 90%, both before  and after loading  as can be observed in The percentages of continuous margins for FP, FRC, and RC at the When performing the fracture strength test on loaded specimens, all prostheses made of fiber reinforced composite (FRC) detached from the abutment before the fracture ocurred. Therefore, no fracture strength data could be procured from this group. A detailed observation of the inlays intaglio surface revealed that the luting composite remained attached to the inlay abutment and that detachments occurred principally between the luting composite and the tooth substrate. Regarding the other two materials, a higher fracture strength was reported for feldspathic porcelain (196N) in respect to Composite Resin (114.9N), as detailed in In respect to failure patterns, half of the prostheses made of feldspathic porcelain (FP) detached from the abutments. Adhesive failures were located between luting composite and tooth substrate and in some cases, remnants of enamel structure were still attached to the prosthesis. The other half failed due to cohesive fractures in the connector's area. All prostheses made of composite resin (CR) failed due to cohesive fractures within the restorative material, mainly located in the connectors' area.The results of the current study could reject the null hypothesis investigated. Marginal adaptation and fracture strength test could identify differences in the fatigue behavior of 2-unit cantilever FPDs made of resin composite, fiber-reinforced composite and feldspathic porcelain.Two-unit resin-bonded cantilever bridges have been used for the replacement of a single missing anterior teeth –5. Compain vitro study, one-abutment inlay retained FPDs were evaluated for their fatigue resistance and fracture strength, to see if increasing the adhesive surface (inlays instead of slots) and limiting the number of retainers to one, could improve their mechanical performance. Feldspathic porcelain and microfilled composite-resin blocks were selected in the present study in agreement with a previous study that employed both materials for the production of CAD/CAM-generated slot-inlay FPDs [In terms of the construction technique and materials' microstructure, the use of machinable composite-resin and/or feldspathic porcelain may be appealing for the construction of adhesive FPDs. The construction of milled FPDs from prefabricated blocks is not only faster, but material quality contributes to better long-term performance. A recent study demonstrated that in three-unit slot-retained FPDs fabricated from composite-resin and glass ceramic blocks, prosthesis fractures and debonding from the abutments led to a high percentage of failures . To overlay FPDs . The thilay FPDs , 7. Threlay FPDs . In addilay FPDs  evaluatelay FPDs  evaluatelay FPDs  assessedcomposite resin-inlay interface showed that a high quality of bonding was achieved between prosthesis material and composite resin used as luting agent  demonstrated the highest marginal adaptation when compared with the other two groups  were used to load the FPDs. Under these experimental conditions, the highest results of fracture strength were attained by feldspathic porcelain (FP), as observed in During the fracture strength test materials fractures or detachments from the abutments occurred at a force of around 20 Kg (196N), which corresponds to a “light” chewing force in the clinical situation. A recent report stated that the mean values for the maximum bite force during mastication varied from 216 to 847N and that posterior fixed partial dentures should withstand loads of at least 500N . In thisFractures of FPDs made of resin composite occurred at a force of 114.9N (around 11 kg), which corresponds to a low mastication force. Such behaviour was expected due to the low fracture toughness of composite resin material and its poor ability to resist the propagation of cracks . TherefoThe fracture strength values of FPDs made of FRC could not be determined since they detached from the abutments prior to fracturing. Detachments occurred between the luting composite and the tooth substrate, suggesting that adhesion to the inlays intaglio surface was not the weak link. Problems related to the limited adhesive surface provided by the MO preparation may explain these detachments. A similar evaluation has been recently performed with three unit inlay-retained FPDs made of the same fiber reinforced material as the one used in this study; their fracture strength was 1373.4N after thermal mechanical stressing . Howeverin vitro study the following conclusions were drawn.Within the limitations of this The null hypothesis was rejected as both, the evaluation of marginal adaptation after thermo mechanical loading and fracture strength testing were able to identify differences in the fatigue behavior of 2-unit cantilever FPDs made of resin composite, fiber-reinforced composite and feldspathic porcelain.The marginal adaptation of feldspathic porcelain, the stiffest material, was comparable to the one of fiber-reinforced composite resin (FRC) . Composite resin (CR) FPDs produced the poorest marginal adaptation. The highest fracture strength was attained by FPDs made of feldspathic porcelain and the lowest by FPDs made of composite resin. All composite resin FPDs fractured due to cohesive failures within the material, suggesting that the material was not sufficiently strong for this application. Fiber reinforced composite FPDs detached from the abutments before they fractured, suggesting that the adhesive surface was insufficient. In respect to feldspathic porcelain, both cohesive and adhesive failures at the luting composite—tooth interface were observed. Further evaluations with an increased abutment preparation and with a core reinforcement are necessary.


The chemokine CXCL13 is known to dictate homing and motility of B cells in lymphoid tissue and has been implicated in the formation of ectopic lymphoid tissue in chronic inflammation. Whether it influences B cell trafficking during acute infection, is largely unclear. In previous studies, we showed that (I) CXCL13 levels are markedly increased in the B cell-rich cerebrospinal fluid (CSF) of patients with acute Lyme neuroborreliosis (LNB), and (II) CXCL13 is released by monocytes upon recognition of borrelial outer surface proteins by Toll-like receptor 2. Here, we assessed the role of CXCL13 - in comparison to other chemokines - in the recruitment of B cells to the CSF of patients with acute LNB.Measurement of chemokines was done by ELISA. B cells were isolated from whole blood using magnetic cell separation (MACS). For migration experiments, a modified Boyden chamber assay was used and the migrated B cells were further analysed by FACS. The migration was inhibited either by preincubation of the CSF samples with neutralizing antibodies, heating to 60°C, removal of proteins >3 kDa, or by pre-treatment of the B cells with pertussis toxin. The principal statistical tests used were one-way analysis of variance and Bonferroni test (chemokine measurements) as well as paired Student's t-test (migration experiments).Measurements of chemokine levels revealed an increase in three of the four known major B cell chemoattractants CXCL13, CCL19 and CXCL12 in LNB CSF. The CXCL13 CSF:serum ratio, as a measure of the chemotactic gradient, was substantially higher than that of CCL19 and CXCL12. Moreover, the chemotactic activity of LNB CSF was reduced up to 56% after preincubation with a neutralizing CXCL13 antibody, while combined preincubation with antibodies against CXCL13, CCL19, and CXCL12 did not lead to further reduction. Since treatment with pertussis toxin, heating to 60°C, and removal of proteins >3 kDa abrogated the chemotactic activity, further not yet identified chemokines seem to be involved in B cell recruitment to LNB CSF.Combined, our study suggests a key role of CXCL13 in B cell migration to sites of infection as shown here for the CSF of LNB patients. Helicobacter pylori gastritis  cells/μl, and the mean CSF/serum albumin ratio was 8.7 [3.3-31.0]. The mean age was 64.7 [48-74] years. Three of these patients suffered from chronic pain, two others had an epileptic seizure, and the further patients were diagnosed amyotrophic lateral sclerosis, vascular compression of the brain stem, Parkinson's disease, transverse myelitis, Guillain-Barré syndrome, multiple sclerosis, herniated vertebral disc, somatoform disorder or progressive ataxia.B.b.-specific antibody production. Detailed data concerning basic CSF findings as well as the B.b.-specific antibody index and the percentage of plasma cells are given in table (ii) 18 patients  with acute (duration of symptoms < 6 months) Lyme neuroborreliosis (LNB) before initiation of therapy. The mean cell count was 146 [1-600] cells/μl  and an elevated CSF white blood cell count according to the criteria described by Marra et al. [(iii) 14 patients  with neurosyphilis (NS). The mean cell count was 17.4 [5-45] cells/μl and the mean CSF/serum albumin ratio was 5.9 [3.2-12.5]. The mean age was 31.8 [19-48] years. The diagnosis was based on serological proof of a syphilitic infection  with polycarbonate membrane filters (5 μm pore). 30 μl of the CSF samples were added to the lower chamber and 50 μl B cell solution was added onto the membrane. The chamber was incubated for 1 h at 37° in humidified air with 5% COFor better comparison of the results from different experiments, a migration index was calculated. We used medium as negative control value and 500 ng/ml rhCXCL13  as a positive control.The results are presented in per cent. A MI of 100% implicates, that the sample had the same chemotactic effect as 500 ng rhCXCL13.6/ml) of isolated B cells and either B cells, which did migrate to rhCXCL13 or LNB CSF, and those that have not migrated were incubated with 5 μl of CD19 and 10 μl of CD27 each for 15 minutes at room temperature in the dark. 1 ml PeliLyse lysing reagent  was added to each of the tubes and incubated for another 10 minutes in the dark at room temperature. The cells were washed and resuspended in 200 μl PBS  and afterwards immediately analysed.Anti-human monoclonal antibodies against CD19  and CD27 , obtained by Immunotech  were used. For flow cytometry 50 μl cell suspension  using FACSDiva software. 10,000 events were acquired. Data were displayed as two colour plots (CD19/CD27). The data of migrated B cells were compared with those of non migrated B cells.From the CSF/serum samples used for the migration assays, sufficient volume for the quantitative measurement of the chemokines CXCL12, CCL19 and CCL21 was available from 12 NIND , 14 NB  and all NS patients. CXCL13 levels in CSF and serum samples were measured in all samples used in the migration assays. A part of the samples have already been analysed for CXCL13 before ,16. The To investigate the chemotactic impact of CXCL13, CXCL12 and CCL19 on the human B cells, the respective chemokines in the CSF samples from LNB patients were blocked with neutralizing antibodies. For the isolated blockade of CXCL13 activity, 10 μg/ml of the neutralizing polyclonal CXCL13 antibody or an isotype control antibody (10 μg/ml)  was used. For triple inhibition assays, we added additional 10 μg/ml polyclonal CCL19 and 50 μg/ml polyclonal CXCL12 antibody or analogous amounts of isotype control antibodies. In pre-tests, we could show that the CXCL13 and CCL19 antibodies inhibit >99% of the chemotactic effect of 50 ng/ml rhCXCL13 and rhCCL19, respectively. In contrast, though testing several available neutralizing antibodies in different concentrations, the blockade of CXCL12 activity only reached 42%. Cross reactivity between all applied antibodies was tested and not observed. All samples were preincubated on a shaker with the respective antibody for 30 min at RT. As negative control, we used a mixture from 12 CSF samples from NIND patients as they would represent the baseline of migratory activity in vivo, 500 ng/ml rhCXCL13 served as positive control. To perform the assay, sufficient CSF volume from 12 patients was available, but only 9 samples were tested as their MI was higher than all NIND samples. In addition, 3 LNB CSF samples were either heated at 60°C for 10 minutes to denature protein structures, filtrated for 90 min at 14 g using a membrane with a 3 kDa Nominal Molecular Weight Limit (Millipore), or the B cells were preincubated for 90 min with 5 μg/ml PTx. All experiments were performed in duplicate.In order to assess statistical significant differences of the chemotactic activity of CSF samples obtained from patients with NIND, LNB, or NS on isolated B cells we used one way analysis of variance (ANOVA) and Bonferroni adjustment. This statistical test was also used to measure the significant differences between the chemokine concentrations of these groups in the ELISA assays. The paired t-test was used to measure the differences in the migration behaviour of B cells between antibody- and isobody-control groups in the inhibition assays. The data were expressed as mean ± SD.First, we determined the chemotactic impact of recombinant human (rh)CXCL13 on human B cells in a well established chemotaxis assay, the modified boyden chamber ,20. rhCXNext, we analysed the migratory activity of CSF samples from different patient groups  on B cells. The migratory index , was clearly higher for the LNB group (MI = 116) than for both other patient groups  Fig. . SimilarTo identify the immune factors responsible for the increased chemotactic activity of CSF samples from LNB patients, we measured the concentration of the four major B cell attracting chemokines CXCL13, CCL19, CXCL12 and CCL21 in CSF samples from all three patient groups . The concentration of CXCL13 was significantly increased in the CSF of LNB patients  compared to NIND  and NS patients  antibodies together. Compared with the isotype controls, blocking the CXCL13 activity alone reduces the percentage of migrated cells up to 56%  Fig. . In moreBordetella pertussis toxin (PTx) to exclude signalling via Gi-proteins . While The mechanisms underlying the B cell rich CSF pleocytosis in LNB are still undefined. Here we demonstrate that CSF of LNB patients is chemotactic to peripheral blood human B cells, and that CXCL13 is a major regulator of B cell recruitment in acute LNB.in vitro setting were predominantly CD27+ B cells. The surface marker CD27+ on B cells indicates, that these cells are activated and can produce 5- to 100-fold more immunoglobulins than CD27- cells [B.b. meningoradiculitis revealed, that large numbers of B cells in the CSF and a prominent intrathecal production of antigens are characteristic for LNB [+) mature type. The exact rate of CD27+ cells in the CSF in LNB, however, has not been determined so far. Results from patients with other neuroinflammatory disorders like multiple sclerosis, viral infections, or neurosyphilis showed that 85% of the total B cell population in the CSF are CD27+, in contrast to 31% in the peripheral blood [+ B cells appear to be the main migrating B cell population in neuroinflammation, both in vivo and in our in vitro experimental setup. Therefore, our in vitro model seems to be a suitable tool to search for the responsible chemotactic agent for B cell immigration in LNB.The cells that migrated towards the CSF of LNB patients in our 7- cells . Earlier for LNB , suggestal blood . Taken tThis chemotactic agent should fulfil four criteria: a) it has to attract B cells, b) it has to be elevated in the CSF of LNB patients and there must be a gradient over the blood-CSF barrier, c) there should be an association between the gradient and the intensity of B-cell-migration, and d) its neutralization should abrogate the observed B cell migration to LNB CSF. The four known major B cell attracting chemokines are CCL19, CCL21, CXCL12, and CXCL13, as shown by Brandes et al. . From reAll of the LNB patients had not been treated with antibiotics before the lumbar puncture. One of the NS patients received an antibiotic therapy for two weeks before CSF sampling and showed the lowest CXCL13 CSF value of all NS patients. This observation fits perfectly to recent studies demonstrating that (I) viable spirochetes are needed for the production of CXCL13 in monocytic cells  and (II)The difference between the patient groups is less impressive for CCL19 but still compatible with a functional chemotactic role: The mean CSF concentration is 8 fold higher than in NIND patients and the mean CSR equals 5.The results obtained for CXCL12 are lower than those from Pashenkov et al. , but theCCL19 instead, which signals via the CCR7 receptor, appears to play a functional role for B-cells only in single cases (as seen in patient Nr. 3, Fig. For the association analysis as shown in Figure B.b. can induce the production of complement in vitro and in vivo [As the inhibition of CXCL13, CCL19 and CXCL12 does not completely abrogate the chemotactic activity of LNB CSF samples, additional factors have to be involved. In a study by Dubois et al., the supernatant of cultured dendritic cells was found to be chemotactic for B cells . As the  in vivo ,34 and t in vivo . However in vivo . Taken tIn summary, CXCL13 plays a key role for the immigration of B cells into the CSF in LNB. Therefore - apart from its unquestioned role in dictating homing and motility of lymphocytes in lymphoid tissues  - this pThe authors declare that they have no competing interests.TAR and UK have conceived of the study, TAR has established the experimental setting and written the manuscript, AP has carried out most of the experiments, MA has done the FACS analysis, MW and SK have collected the CSF and serum samples, CS and MK have participated in the design of the study and the statistical analysis and helped drafting the manuscript. HWP and UK have participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.


The conference centered on chronic intracranial hypertension (IH) including the latest research and clinical information. It was sponsored by the Intracranial Hypertension Research Foundation and held at the University of Texas Medical School, Houston, on June 21–22 The Intracranial Hypertension Research Foundation (IHRF) in Vancouver, Washington, promotes research into the pathophysiological basis of chronic IH and the evolution of better treatment and, ultimately, a cure. IHRF is a multi-functional organization. It not only encourages research, but also facilitates understanding and management of chronic primary and secondary IH, through research, training and education programs worldwide.IHRF sponsors programs for researchers and clinicians, as well as educational conferences for patients and families. The Houston conference was attended by 108 patients and family members who heard presentations by nine clinicians and researchers.Patients asked many questions during extensive panel sessions. The conference also allowed patients to speak about personal experiences with the disorder. IHRF held the first conference on chronic IH in 2006 at the Oregon Health & Science University (OHSU). A wide variety of subjects were presented to enhance patient knowledge and awareness. Patient education importantly improves patient insight about the disorder. It also facilitates active cooperation with their physicians. Understanding limitations of accepted medical and surgical treatment leads to realistic goals in management.Additionally, patient education is vital in controlling health care costs.The opening presentation by Conrad Johanson, Professor of Clinical Neuroscience at Brown University dealt with CSF production in the choroid plexus and its possible role in IH. Covered in the lecture was the structure of the choroid plexus, the dynamic turnover of ions and water in CSF production by the choroidal epithelium, the transport of CSF solute for the brain, and homeostasis of CNS extracellular fluid. A discussion of translational research goals involving the choroid plexus and CSF and possible directions for research in IH concluded the presentation.John McGregor, Associate Professor of Neurosurgery at Ohio State University, spoke on "Neural Hydrodynamics Disorders: Hydrocephalus, Intracranial Hypertension and the Chiari Malformation. Relationships, Similarities and Differences" which was followed by two lectures covering CSF shunt and valve technology. The latter two presentations discussed in depth the available technology with details of the features of each device.The conclusion was that despite the numerous designs, the overall success rate for the various shunts and valve equipment are similar. Better designed shunts are greatly needed.The next lecture by Conrad Johanson: "Current Theories on Causation and Reduction of Elevated CSF Pressures: Implications for Intracranial Hypertension", began with a discussion of CSF fluid dynamics centering on CSF reabsorption including the controversial arachnoid and CSF lymphatic drainage. The role of neuropeptides such as atrial natriuretic peptide (ANP) in CSF production and the role of growth factors like basic fibroblast growth factor (FGF2) in CSF reabsorption, were identified. The intriguing possibility of peptides as pharmacological agents in controlling intracranial pressure (ICP) was thought provoking and stimulating, especially since no specific drug is available to control production and egress of CSF.Steven Katz, neuro-ophthalmologist and Associate Professor of Ophthalmology at Ohio State University, lectured on the symptoms and signs in idiopathic intracranial hypertension (IIH) and the medical management options. He discussed approaches that work best in his practice. Steven Katz had previously demonstrated the somatostatin receptors 1 and 2 in normal human choroid plexus and arachnoid granulations and thus surmised that somatostatin is involved in CSF production and egress. His preliminary clinical use of octreotide, a peptide that mimics somatostatin, was discussed, including the potential complications of somatosatin analogs to control IH. Steven Katz described his techniques for optic nerve sheath decompression and demonstrated findings from many of his procedures. He attributed excellent outcomes to reduced surgery duration, i.e. especially the time of optic nerve stretching. He emphasized that short exposure and optimal surgical approach lead to minimal diplopia, ptosis and most importantly, minimal vision loss.Marc Criden, neuro-ophthalmologist and Assistant Professor of Ophthalmology at the University of Texas, Houston, presented an updated overview of pediatric IH. In children under 10 years, gender and weight are not the factors they are in adults. He pointed out that many physicians consider IH in children under 10 to be a different disorder because of these characteristic differences. Headaches associated with IH and how best to manage them were discussed by neurologist and neuro-ophthalmologist, Leonard Hershkowitz of Baylor University. He indicated his management techniques that worked well for him.A reception followed in which patients again communicated with the speakers.The 2nd conference day opened with a discussion of the mission and goals of IHRF and a review of IHRF-sponsored research by Emanuel Tanne, Clinical Assistant Professor of Ophthalmology, OHSU and president of IHRF. He pointed out that IHRF works to remove significant obstacles to research: under-funding, under-coordination of effort, incomplete recognition of the life-altering effects of IIH, and low recruitment of researchers in this area. IHRF funded animal model development projects at the University of Chicago and the University of Arkansas. Under investigation is a knockout mouse that develops IH shortly after weaning. Other research areas included joint testing at the University of Utah of a NASA-developed, non-invasive, closed loop ultrasound device to measure ICP in microgravity and an investigation of vitamin A receptors in arachnoid granulations at Ohio State University. IHRF also partners with the Casey EyeInstitute, in regard to the Intracranial Hypertension Registry at the Oregon Health & Science University, Portland. The IH Registry is a relational database management system designed for medical research. Emanuel Tanne discussed ongoing Registry research, including studies in genetics, economics, weight gain and pregnancy. Jessica Tanne, IHRF Director, Communications & Development, discussed raising community awareness, fundraising and the importance of becoming an IH ambassador. Illustrating and discussing their fundraising creativity were Dori Clements and Jacque Tate, both parents of IH children.Clark Sitton, Assistant Professor of Radiology, University of Texas, Houston, discussed types of imaging studies and the goals of imaging in IH. He presented an extensive collection of studies demonstrating pathological findings associated with IH.Bariatric surgery as a possible option in the management of IH was presented by Erik Wilson, Assistant Professor of Surgery, University of Texas, Houston, who covered risks and benefits of bariatric surgery, using his extensive experience as a guide. He also discussed types of procedures available and his personal approach to follow-up and long term goals for patients. Marc Criden presented a new hypothesis to consider for clinical management of chronic IH: establishing target pressures for each patient with IH. The hypothesis arose out of his and Steven Katz's experience with patients at the last IHRF patient conference, where they were impressed by the variety of symptoms and patient reports contradictory to current teaching. They hypothesized that chronically elevated ICP is neurologically damaging, even in patients without significant visual dysfunction or intractable headache. They are presently investigating and evaluating this hypothesis. The final speaker, Kapil Kapoor, resident in ophthalmology at the University of Texas, Galveston, presented his research on hyposmia and IH. He found that patients with elevated ICP have a decreased sense of smell and concluded that other nerves such as the olfactory nerve may be functionally compromised in the setting of IH by a mechanism similar to that of optic nerve compression. Therefore, it may be appropriate to consider IH as a more global neuroanatomic insult of augmented CSF pressure than previously considered.While the intent of educating patients was admirably accomplished by this conference, the conference served as a unique experience for researchers and clinicians to hear from a large group of patients about the nature of their disorders. Therefore, not only did physicians from a variety of sub-specialties have an opportunity to exchange ideas and explore future collaborative projects they also left with a different perspective of chronic IH and a new appreciation of patient-centered conferences as a mechanism to expedite translational CSF research.The author declares he has no competing interests.I am the sole author and have read and approved the final version of this paper.


Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of β-amyloid peptide (Aβ) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of Aβ (Aβ42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood.  Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why Aβ or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in Aβ or tau levels, making it possible to detect subtle alterations over time. In combination with Aβ and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF Aβ42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF Aβ or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes. Pulling the glass capillary tube              The glass capillary tube is purchased from The Sutter Instrument Inc . Pull the capillary tubes on a Sutter P-87 Flaming micropipette puller, with the heat index set at 300 and the pressure index set at 330. Trim the tip of the glass capillary tube with scissors, so that the tapered tip has an inner diameter of about 0.5 mm.Cisterna magna puncture technique for CSF              1.Figure 1CSF samples are taken from the cisterna magna  using a method that was published previously oC incubator.1. The mice are anesthesized by Ketamine (100mg/kg) and xylazine (10mg/kg), administered intraperitoneally. During the time of anesthesia induction, the mice are kept in a 37iodine, followed by 70% ethanol (repeat 3 times), and a sagittal incision of the skin is made inferior to the occiput.2. The skin of the neck is shaved, and the mouse is then placed prone on the stereotaxic instrument with direct contact of a heating pad. A rectal temperature probe is inserted into the rectum so that the heat produced by the heating pad is adjusted in response to the changes of body temperature. The head is secured with the head adaptors. The surgical site is swabbed with 10% povidone m. biventer cervicis and m. rectus capitis dorsalis major) are separated by blunt dissection with forceps. A pair of microretractors is used to hold the muscles apart.3. Under the dissection microscope, the subcutaneous tissue and muscles , and the CSF space are visible .6. The dura mater is blotted dry with sterile cotton swab. Penetrate the capillary tube into the citerna magna through the dura mater, lateral to the arteria dorsalis spinalis . Following a noticeable change in resistance to the capillary tube insertion, the CSF flows into the capillary tube.7. Carefully remove the capillary tube, and connect it to a 3 ml syringe through a polyethylene tubing that has a 1 mm internal diameter. Inject the CSF into a pre-marked 0.5 ml eppendorf tube, and freeze the tube immediately on dry ice and then transfer it into a -80°C freezer.8. After CSF sampling, the muscles are re-aligned, and skin is sutured . About 1 ml of 0.9% NaCl is injected subcutaneously to prevent de-hydration. The mouse is kept at the incubator to maintain body temperature until it recovers; the weight of the mouse is monitored 1 day, and 1 week after the surgery.        Results and Discussion              We described a highly reliable protocol for serial sampling of CSF from mice without detectable plasma contamination.2, the average volume is about 5 μl (3 to 7 μl), while the P301L (JNPL3) mice 3 give a yield of about 10 μl- 15 μl. For serial sampling, a maximum of 7-8 μl can be safely taken each time at an interval of 2-3 months.1. The whole procedure usually takes 10 min per mouse (including anesthesia). The volume of CSF obtained is dependent on the mouse strains. In the PS/APP double Tg mice we used 4.2. During the surgery, it's important to position the head and the body of the mouse properly on the stereotaxic frame so that the dura mater of cisterna magna can be exposed sufficiently. Carefully avoid the blood vessels when penetrating the dura mater with the capillary tube to prevent contamination from plasma proteins, which can be monitored in the CSF sample by immunoblotting for ApoB 3. Minimizing the tissue damage during each surgery is important, as tissue conglutination can increase the difficulty by predisposing bleeding for the following sampling.5.4. The body temperature needs to be well maintained during the surgery because hypothermia caused by anesthesia can significantly affect the phosphorylated tau levels in the brain very rapidly 6. Two μl of CSF from the PS/APP mice will give satisfactory results when diluted 1:50-1:60. For the tau ELISA, 4-5 μl CSF from P301L (JNPL3) mice will be sufficient for the detection of total tau, or tauphosphorylated at threonine231 (Kits are purchased from Bio-Source).5. The Ab ELISA protocol and antibodies that we used were described in detail in Refolo et al. 6. This technique can be applied to other mouse models of neurological diseases, for which a small volume of CSF is satisfactory for desired assays.We described a highly reliable protocol for serial sampling of CSF from mice without detectable plasma contamination.1. The whole procedure usually takes 10 min per mouse (including anesthesia).  The volume of CSF obtained is dependent on the mouse strains. In the PS/APP double Tg mice we used2, the average volume is about 5 μl (3 to 7 μl), while the P301L (JNPL3) mice 3 give a yield of about 10 μl- 15 μl.  For serial sampling, a maximum of 7-8 μl can be safely taken each time at an interval of 2-3 months.4.2. During the surgery, it is important to position the head and the body of the mouse properly on the stereotaxic frame so that the dura mater of cisterna magna can be exposed sufficiently. Carefully avoid the blood vessels when penetrating the dura mater with the capillary tube to prevent contamination from plasma proteins, which can be monitored in the CSF sample by immunoblotting for ApoB 3. Minimizing the tissue damage during each surgery is important, as tissue conglutination can increase the difficulty by predisposing bleeding for the following sampling.5.4. The body temperature needs to be well maintained during the surgery because hypothermia caused by anesthesia can significantly affect the phosphorylated tau levels in the brain very rapidly 6. Two μl of CSF from the PS/APP mice will give satisfactory results when diluted 1:50-1:60. For the tau ELISA, 4-5 μl CSF from P301L (JNPL3) mice will be sufficient for the detection of total tau, or tau phosphorylated at threonine 231 (Kits are purchased from Bio-Source).5. The Ab ELISA protocol and antibodies that we used were described in detail in Refolo et al. 6. This technique can be applied to other mouse models of neurological diseases, for which a small volume of CSF is satisfactory for desired assays.


A 41-year-old female patient was admitted with streptococcal meningitis on a background of 5-month history of CSF rhinorrhoea. Imaging revealed an extensive skull base lesion involving the sphenoid and ethmoid sinuses, the pituitary fossa with suprasellar extension and bony destruction. Histological examination of an endonasal transethmoidal biopsy suggested a diagnosis of olfactory neuroblastoma. A profuse CSF leak occurred and the patient developed coliform meningitis. A second endonasal endoscopic biopsy was undertaken which demonstrated the tumour to be a prolactinoma. Following endonasal repair of the CSF leak and lumbar drainage, she developed profound pneumocephalus. The patient underwent three further unsuccessful CSF leak repairs. Definitive control of the CSF leak was finally achieved through a transcranial approach with prolonged lumbar drainage. This case illustrates some of the potentially devastating complications which can occur as a consequence of complex skull base lesions. A multidisciplinary approach may be required to successfully manage such cases. Traditionally, complex paranasal and anterior skull base lesions have been managed with either transcranial or transphenoidal microsurgical approaches. The application of endoscopy to neurological surgery and advances in neuronavigation have provided further options to skull base surgeons. One of the commonest complications of skull base lesions and their management is the occurrence of CSF leakage, which can result in the potentially fatal consequences of pneumocephalus and meningitis. Despite advances in microsurgical and endoscopic technology and expertise, definitive management of complex lesions and their resultant complications may require multidisciplinary team management and open transcranial neurosurgery. A 41-year-old female patient was referred to the ENT outpatient clinic with 5-month history of clear fluid discharging from the nose. Fluid samples were sent for tau protein analysis and outpatient imaging was requested. Prior to completion of skull base imaging, the patient was admitted in an acute confusional state, with a nonblanching rash, pyrexia, and signs of meningism. A diagnosis of streptococcal meningitis was made following lumbar puncture and she was commenced on appropriate antibiotics. Magnetic resonance imaging demonstrated an extensive skull base lesion involving the sphenoid and ethmoid sinuses, pituitary fossa, and suprasellar region . ComputeAn endonasal transethmoidal biopsy was undertaken, following which there was profuse CSF rhinorrhoea. Subsequently, the patient developed clinical signs of meningism with pyrexia, neck stiffness, photophobia, and deterioration in neurological status. CSF analysis revealed gram-negative coliforms and antibiotic treatment was commenced.The patient was transferred to the regional neuroscience centre for joint neurosurgical and ENT management. On recovery from the second episode of meningitis, she underwent an endoscopic endonasal biopsy and repair of the anterior pituitary fossa and planum sphenoidale using layered fat graft and artificial dural substitute sealed with Tisseel fibrin sealant . Histological analysis of this biopsy specimen confirmed the tumour to be a prolactinoma. Although her initial serum prolactin level was only 451 miu/mL , it did rise to 953 miu/mL in the immediate postbiopsy period. Following endocrine review, she was commenced on cabergoline.Despite satisfactory intraoperative appearances, CSF rhinorrhoea recurred. A CT cisternogram was undertaken to further characterise the site of CSF leakage . ThroughBoth attempts were unsuccessful and lumbar CSF drainage resulted in profound pneumocephalus . The patOnce sufficient neurological recovery had occurred, a second CT cisternogram  was undeFollowing the fourth unsuccessful attempt, the patient underwent a transcranial repair of the CSF leak through a right-sided pterional craniotomy. Intraoperatively, no dural defect was visible; however, bony defects in the anterior pituitary fossa floor were palpable and therefore sealed with layers of temporalis fascia and muscle grafts secured with Tisseel sealant. A prolonged post-operative period of lumbar CSF drainage (7 days) was also undertaken which resulted in a successful control of CSF rhinorrhoea. The initial and subsequent biopsies were reviewed and showed an identical tumour consisting of sheets of cells with mildly pleomorphic round to oval nuclei and moderate amounts of eosinophilic cytoplasm . MitoticThe difficulties encountered in definitive occlusion of the bony defects resulting in CSF rhinorrhoea in this case are likely to have resulted from a number of causes. Whilst bony destruction is a recognised feature of aggressive prolactinomas, the extensive infrasellar growth pattern seen in this case is unusual . Once thThe potential pitfalls in differentiating olfactory neuroblastoma from prolactinoma and other paranasal tumours on histological examination have previously been described . The hisComplex paranasal and skull base lesions can result in bony defects and problematic CSF leaks. Although dopamine receptor agonists are the treatment of choice for prolactinomas, tumour shrinkage can take several weeks and residual tumour may contribute to recurrence of CSF leakage. Consequently, tumour debulking might be considered in patients with recurrent CSF rhinorrhoea in whom a histological diagnosis of prolactinoma has been confirmed. This case illustrates some of the potentially devastating complications of untreated CSF rhinorrhoea including recurrent meningitis and consequent raised CSF pressure, and pneumocephalus resulting in seizures and reduced conscious level. Management of these lesions requires a multidisciplinary approach involving ENT surgeons, neurosurgeons, neuroradiologists, and endocrinologists. The time at which endoscopic or transphenoidal approaches are abandoned in favour of transcranial surgery will depend on the surgical experience of the team and the anatomy of the site of CSF leakage. Despite advances in neuro-navigation and endoscopic surgery, transcranial repair maybe the only successful solution.


We report acute encephalopathy associated with influenza A infection in 3 adults. We detected high cerebrospinal fluid (CSF) and plasma concentrations of CXCL8/IL-8 and CCL2/MCP-1 (CSF/plasma ratios >3), and interleukin-6, CXCL10/IP-10, but no evidence of viral neuroinvasion. Patients recovered without sequelae. Hyperactivated cytokine response may play a role in pathogenesis. Altered consciousness, disorientation, and seizures occur within a few days after the onset of fever and respiratory symptoms  have been described  (7). In Concentrations of oseltamivir phosphate (OP) and its biologically active metabolite oseltamivir carboxylate (OC) were measured in CSF and plasma taken simultaneously from 1 patient who received concurrent treatment, using tandem mass spectrometry .The clinical and virologic findings are summarized in ,Despite apparently normal CSF findings, high concentrations of cytokines/chemokines were detected in the CSF and plasma specimens of all patients . Plasma Simultaneous CSF and plasma OC and OP concentrations were determined for patient 3, as symptoms progressed at 18 h after oseltamivir. The concentrations (mean ± SD) of OC in duplicate CSF and plasma samples were 18.3 ± 0.9 ng/mL and 143.8 ± 3.3 ng/mL, respectively; the CSF/plasma concentration ratio was 12%–13%. The OP plasma concentration was 1.05 ± 0.03 ng/mL; it was not detectable in the CSF.,We report 3 adults with acute encephalopathy  associated with influenza. High CSF and blood cytokine/chemokine  levels were detected. No evidence of direct viral neuroinvasion was found. All patients recovered rapidly without sequelae  was detected in the CSF of patient 3; the CSF/plasma concentration ratio was 12%–13% (18.3/143.8 ng/mL) at 18-hours postdose. This degree of CSF penetration is similar to that observed among healthy patients, with a Cmax CSF/plasma concentration ratio of 3.5% (at ≈8 hours), and a ratio of ≈10% at 18 hours (concentration-time profiles for plasma/CSF differ). Assuming a similar ratio, the CSF OP concentration would have fallen below the assay’s detection limit (0.25 ng/mL) by 18 hours (,Our study is limited by the small patient number and the lack of feasibility in obtaining CSF for study/comparison in influenza patients without neurologic symptoms. Further studies on the clinical spectrum of influenza encephalopathy and encephalitis in adults (Acute Encephalopathy Associated with Influenza A Infection in Adults


Cerebrospinal fluid (CSF) sodium levels have been reported to rise during episodic migraine. Since migraine frequently starts in early morning or late afternoon, we hypothesized that natural sodium chronobiology may predispose susceptible persons when extracellular CSF sodium increases. Since no mammalian brain sodium rhythms are known, we designed a study of healthy humans to test if cation rhythms exist in CSF.Lumbar CSF was collected every ten minutes at 0.1 mL/min for 24 h from six healthy participants. CSF sodium and potassium concentrations were measured by ion chromatography, total protein by fluorescent spectrometry, and osmolarity by freezing point depression. We analyzed cation and protein distributions over the 24 h period and spectral and permutation tests to identify significant rhythms. We applied the False Discovery Rate method to adjust significance levels for multiple tests and Spearman correlations to compare sodium fluctuations with potassium, protein, and osmolarity.The distribution of sodium varied much more than potassium, and there were statistically significant rhythms at 12 and 1.65 h periods. Curve fitting to the average time course of the mean sodium of all six subjects revealed the lowest sodium levels at 03.20 h and highest at 08.00 h, a second nadir at 09.50 h and a second peak at 18.10 h. Sodium levels were not correlated with potassium or protein concentration, or with osmolarity.These CSF rhythms are the first reports of sodium chronobiology in the human nervous system. The results are consistent with our hypothesis that rising levels of extracellular sodium may contribute to the timing of migraine onset. The physiological importance of sodium in the nervous system suggests that these rhythms may have additional repercussions on ultradian functions. Many behaviorally related dynamical processes in the nervous system are characterized by episodes of complex oscillatory states whose periodicity may be expressed over multiple temporal and spatial scales . Mood 22 and man+]csf) increased during migraine while blood plasma sodium concentration did not change [+]csf is rapidly equilibrated with extracellular sodium concentration ([Na+]e) [+]csf in migraine based on increased activity of capillary endothelial cell sodium potassium ATPase (NKAT) [+]csf/e may contribute to the triggering of migraine.Migraine commonly has an onset in early morning or late afternoon, suggesting an underlying biochemical rhythm may predispose to this temporal variability -11. We dt change . [Na+]cs([Na+]e) -15 and we (NKAT) . Many ote (NKAT) . Many lie (NKAT) . Whateve+]csf/e chronobiology, we designed experiments in nonheadache-suffering volunteers to investigate whether there are any physiological rhythms of sodium in CSF. We predicted that a rising [Na+]csf in the early morning and late afternoon would be a feature of normal brain sodium homeostasis if [Na+]csf/e change is relevant to migraine time of onset.Sodium is crucial for neuronal excitability and, since there are no published data on  and [K+]. The instrument is comprised of an IP25 isocratic pump, an EG40 eluent generator with a methyl sulfonic acid cartridge, an Ultra II cation self-regenerating suppressor (CSRS), and a CD 20 conductivity detector employing an un-thermostated conductivity detection cell. Analyses were performed on 4 mm diameter CG12A (50 mm) guardand CA12A (250 mm) analytical ion exchange columns with methysulfonic at 18 mM (isocratic) and a flow rate of 1.0 mL/min. The CSRS was operated at 100 mA. were injected by an AS40 autosampler from 5 mL vials with integral filter caps.CSF samples were thawed and 10 μL was diluted by 1:1,000 CSF with Milli-Q ultra pure de-ionized water , well mixed and transferred into 5 mL PolyVials with Filtercaps . A Dionex AS 40 autosampler was used to transfer samples to a 25 uL sample loop for injection, that were analysed in duplicate on a DIONEX DX-500Ion Chromatography system to determine [Na+] and > 0.995 for [Na+] spanning the diluted concentrations of unknowns. Quality control standards were run every 25 samples to ensure accuracy of analysis (95-105% recovery). Random samples were re-analyzed to check reproducibility. Chromatographic peaks were integrated using DIONEX PeakNet software and results plotted. CSF was run in duplicate and the results averaged for all samples. Any discrepancy between the two duplicate measures was re-analyzed until the deviation was overcome.Sodium and potassium cations were calibrated (and continuously verified) with NIST-certified standard mixtures . Cation calibration curves were linear with correlation coefficients > 0.99 for  and [K+] for all six participants . Nevertheless, the reproducibility of the assays was clearly not limiting, as the SDs of the same CSF run ten times on different days  was much less than the SDs of the 24-h measures. Thus although some [Na+] values are not in the typical range, we have confidence in the reproducibility of the assays.The distributions of the 24 h CSF csf Power Spectral Densities) and 2 .Furthermore to more rigorously test for rhythms in the 24-h CSF spectra, we applied DFT with permutation analysis as described in the Methods. Figure Because of concern that these apparent sodium rhythms may be correlated with changes in osmolarity or total protein, we determined Spearman correlation coefficients with CI. For osmolarity/Na: r = -0.22 . For Na/protein: r = 0.24 . Based on these weak correlations, we can conclude the variables of osmolarity and total protein are not the cause of the sodium fluctuations.Potassium levels had minimal change over 24 h  is sufficient to have physiological effect on neuronal excitability. Moreover, this data raises the possibility that efforts to reduce the natural sodium variation or to administer drugs to coincide with sodium chronobiology may reduce migraine in susceptible people. Hodgkin and Katz [+]e. When a neuron is at rest, the Na+ influx through voltage-gated Na+ channels is low, as these channels are usually closed or inactivated. However, the channel gate is displaced when [Na+]e increases [+]e speeded recovery from the inactivation state, enabling an earlier action potential and leading to hyperexcitability [+]csf caused a sympathetic hyperactivity response (increasing blood pressure and heart rate) through increasing ouabain-like substances and activating the brain renin-angiotensin- aldosterone system csf: sodium concentration in CSF; [Na+]e: sodium concentration in brain extracellular fluid; NKAT: Na+, K+,-ATPase transporter; PSD: power spectral density.CSF: Cerebrospinal fluid; CSRS: cation self-regenerating suppressor; DFT: discrete Fourier transformation; FDR: false discovery rate; csf Power Spectral DensitiesSix individual csf Power Spectral Densities and their AveragePermutation analysis statistics of six individual [Na. Permutation analysis statistics of six individual [Na+]csf Power Spectral Densities and their Average.Click here for file