An Experimental Study of Data Migration Algorithms

. The data migration problem is the problem of computing

Connections of the Tectum of the Rattlesnake Crotalus viridis: An HRP Study

ABSTRACT We have studied the connections of the tectum of the rattlesnake by tectal application of horseradish peroxidase. The tectum receives bilateral input from nucleus lentiformis mesencephali, posterolateral tegmental nuclei, anterior tegmental nuclei and periventricular nuclei; ipsilateral input from nucleus geniculatus pretectalis, and lateral geniculate nucleus pars dorsalis; and contralateral input from dorso-lateral posterior tegmental nucleus and the previously undescribed nucleus reticularis caloris (RC). RC is located on the ventro-lateral surface of the medulla and consists of large cells 25-45 km in diameter. Efferent projections from the tectum can be traced to the ipsilatera1 nucleus lentiformis mesencephali, the ipsilateral lateral geniculate region, anterior tegmental region and a wide bilateral area of the neuropil of the ventral tegmentum and ventral medulla. We have not found any direct tectal projections from the sensory trigeminal nuclei including the nucleus of the lateral descending trigeminal tract (LTTD). We suggest that in the rattlesnake, RC is the intermediate link connecting LTTD to the tectum. Snakes of the subfamily Crotalinae (fam. Viperidae) and of the family Boidae possess infrared-sensitive pit organs which are innervated by branches of the trigeminal nerve (Lynn, '31). These primary fibers project to a nucleus of the bulbar trigeminal sensory complex. This nucleus is called the nucleus of the lateral descending trigeminal tract and designated LTTD by Molenaar ('74, '77). Schroeder and Loop referred to the same nucleus as the nucleus descendens lateralis nervi trigemini, designating it DLV; they provided the first experimental evidence that this nucleus is involved in the infrared sensory system by tracing cobalt, iontophoresed into the superficial maxillary branch and part of the mandibular branch of the trigeminal nerve (Loop and Schroeder, '75; Schroeder and Loop, '76). Infrared-sensitive units, driven by the ipsilateral pit organs, have been recorded in the region of LTTD or its tract in both crotalids (Terashima and Goris, '77; Stanford and Hartline, '78) and boids (Molenaar, '78b We have presumed that the infrared input to the tectum comes by way of the LTTD; a direct projection from LTTD to the tectum in pythons was recently proposed by Molenaar and Fizaan-Oostveen ('78), and in crotalines by Auen ('78). We recognized the need for a thorough investigation of the LTTD-to-tectum pathway in the rattlesnake. Accordingly, we have examined tectal connections in Crotalus using horseradish peroxidase. We report here an important finding of our study: the 31 32 GRUBERG, KICLITER, NEWMAN, KASS AND HARTLINE absence of a direct projection from LTTD to tectum, a t least in the crotaline species we used. At the same time, we describe a nucleus with a tectal projection that is a strong candidate as an infrared system intermediate between LTTD and tectum. We also describe other efferent and afferent connections of the rattlesnake tectum. MATERIALS AND METHODS We used adult southern Pacific rattlesnakes, Crotalus uiridis. Animals were between 40 cm and 120 cm in length. Morphology of brain For cytoarchitectonic observations we prepared stained brains with either a modification of the Davenport et al. Bodian protein silver method (Gray, '75) or with cresyl violet. The brains were embedded in paraffin and cut at 15 pm in the transverse plane. Tectal HRP iontophoresis Preliminary experiments revealed that horseradish peroxidase introduced into the tectum by either hyraulic injection or by application of soaked pledgets would be accumulated by retrograde transport in several extratectal nuclei. In order to make more circumscribed injections, we then used an iontophoretic method previously described (Gruberg and Udin, '78). Seven animals were anesthetized with metofane, their tecta exposed and the dura and arachnoid removed. Tapered glass capillary tubes with tip diameter of 15-20 pm were filled with a freshly prepared 25% solution of HRP (Sigma Type VI) in 0.05 M Tris buffer a t pH 8.6. The capillary tubes were connected to a pulse generator which was adjusted to pass 1 pA at 5 Hz (square waves with 50% duty cycle). The tips of the HRP-filled electrodes were positioned 200-600 pm below the tectal surface by a micromanipulator. Deeper depths were used for more lateral penetrations. Three to four widely separated unilateral tectal injections were made in each animal. Current was passed for five to ten minutes at each site. Following survival times and temperatures ranging from two days at 22°C to nine days at 29"C, the animals were again anesthetized and perfused first with saline and then with 2.5% glutaraldehyde and 2% paraformaldehyde in pH 7.4 phosphate buffer. Their brains were removed from the cranium, fixed an additional three hours and placed overnight in a phosphate buffer with 10% sucrose. On the following day, the brains were frozen and cut at 40 pm in a cryostat at -10°C. Sections were collected on subbed slides and thoroughly dried. They were then treated by the blue benzidinelsodium nitroferricyanide method (Mesulam, '76). RESULTS Wherever possible, we have followed the nomenclature used in cytoarchitectonic studies of previous authors (Warner, '35, '47; Halpern and Frumin, '73; Northcutt and Butler, '74; Molenaar, '77). However, a comprehensive description of the rattlesnake brain has not been made. We have therefore, relied on the terminology used for other reptiles (Northcutt and Butler, '74) and used our best judgment in naming several cell groups. The results of the tectal HRP injection studies are summarized in figure 2, which shows in diagrammatic form the fiber tracts, cells and injection sites stained with HRP reaction product. The injection sites were approximately 400-500 p m in diameter ( Medulla projections The result of most immediate interest to us was that HRP activity was absent in the cells of the LTTD in all our animals. Thus we find no evidence for a direct projection from the LTTD to the tectum in the rattlesnake. In fact, we consistently found that only one circumscribed nucleus in the medulla contralatera1 to the injection sites contained stained cells (figs. 2J-L, 4, 6). This nucleus had not been described in snakes by previous authors. We suggest here that it be named "nucleus reticularis caloris" (RC). RC is made up of large cells approximately 25-45 pm in diameter ( In Bodian-stained material the RC stands out clearly as a collection of transversely planar multipolar cells which are interleaved by large, longitudinally oriented axons of approximately 5 p m diameter ( We found HRP-labelled cells throughout almost the entire length of the RC contralateral to the injected tectal lobe. We saw no stained cells in the ipsilateral RC. All brains showed stained cells in the contralateral RC except for the shortest survival time (2 daysat 22°C). Longer survival times, seven and nine days, gave denser staining than the shorter times. The pathway from RC to tectum is clearly demarcated in our HRP material. Fibers exit the nucleus medially and pass near or through the tbsd, crossing the midline in a tope-shaped bump (figs. 2I,J, 6). They then form a longitudinally oriented tract near the ventral surface about one-third the distance lateral from the midline. The tract proceeds rostrally into the caudal tegmentum where it courses dorsally to the tectum along the lateral surface of the brain. The only other HRP-stained cells in the medulla were scattered in and near the ipsilateral division of the RC-tectal tract ( Other afferent tectal projections 1. Postero-lateral tegmental nucleus (PLT) A group of HRP-stained cells lies in a narrow dorsoventral band near the lateral edge of the caudal tegmentum (figs. lH, 2G). These cells are bilaterally distributed. The contralateral stained cells are more ventrally located than the ipsilateral cells of this nucleus. We can trace a pathway backward from the injected tectum exiting caudal of the third nerve nucleus, running ventrad down the lateral edge of the tegmentum to the ipsilateral postero-lateral tegmental nucleus ( Antero-lateral tegmental nucleus (ALT) A bilaterally stained cell group lies in the rostral tegmentum ( 3. Dorso-lateral posterior tegmental nucleus (DLT) Another group of stained cells, contralateral to the injected tectal lobe, lies a t the extreme caudal level of the dorsal tegmentum ( Pretectal complex A set of cells of nucleus lentiformis mesencephali (LM) ( Hypothalamus Labelled cells are distributed bilaterally in the periventricular nucleus of the hypothalamus (PHI 2. Tectal commissure fibers are sparsely distributed in the intermediate layers of the contralateral tectum (figs. 2F,G). Some of these fibers can be followed through the contralateral tectum laterally and ventrally to the anterior tegmental field. 3. The main descending projections from the tectum exit from an intermediate tectal layer and course ventrally at approximately the level of the third nerve nucleus. Fibers can be distinguished in three tracts. One set of fibers collects on the ipsilateral ventral surface of the tegmentum (tbv). Another set of uncrossed fibers maintains a more dorsal location (tbd). The third set of fibers (tbsd), which seems comparable to the radiations of Meynert in mammals (Kappers et al., '361, crosses and collects in a deep medial tract of the contralateral tegmentum ( DISCUSSION In our preparations, the main cell groups that project to the optic tectum are: the pretectal complex bilaterally; postero-lateral tegmental nuclei bilaterally; lateral geniculate par dorsalis ipsilaterally; anterior tegmental complex bilaterally; dorso-lateral posterior tegmental nucleus contralaterally; periventricular nucleus bilaterally; and the nucleus reticularis caloris contralaterally. Stanford and Schroeder ('79), using FinkHeimer degeneration staining after electrolytic lesions, have demonstrated a direct projection from LTTD to the ipsilateral RC in rattlesnakes. They did not find a direct projection from LTTD to the tectum nor to any other cell group that projects directly to the tectum. Our findings, combined with these degeneration results, suggest that RC is the intermediate link connecting the LTTD to the tectum. It is not clear whether there are structures homologous to RC in other vertebrate species. The RC is closely related to the trigeminal complex given its input from ipsilateral LTTD. It is generally agreed that the primary trigeminal complex in the medulla oblongata of higher vertebrates is divided into two parts -the main (principal) trigeminal nucleus and an elongated descending (spinal) division. The descending division has in turn been divided into three nuclei which are, in rostrocaudal order, the nucleus oralis, the nucleus interpolaris and the nucleus caudalis (Olszewski, '50). The L'M'D in rattlesnakes is located in the caudal end of the medulla and in the rostra1 spinal cord. This location suggests that the 40 GRUBERG, KICLITER, NEWMAN, KASS AND HARTLINE LTTD is related to, and perhaps is a subdivision of the nucleus caudalis seen in mammals (Olszewski, '50). This is supported by an electron microscope study of the closely related pit viper, Agkistrodon (Meszler, '751, which shows that the cellular and synaptic architecture of the LTTD most closely resembles that of the nucleus caudalis. The nucleus caudalis has been shown to project to the ipsilateral reticular formation in a number of mammals (Carpenter and Hanna, '61; Stewart and King, '63; Dunn and Matzke, '68; Kawamura, '71; Roberts and Matzke, '71; Tiwara and King, '74). Additionally, Tiwara and King found a small projection from the lateral reticular formation to the contralatera1 tectum. However, their lesions in the reticular formation were adjacent to the nucleus caudalis and not more rostra1 where the bulk of the nucleus caudalis-to-reticular formation projection lies (Darian-Smith, '73; Carpenter and Hanna, '61; Stewart and King, '63). Thus in mammals no direct projections have been found from the primary trigeminal nuclei to the tectum. There exists, however, an area of the reticular formation which receives trigeminal input and in turn projects to the contralateral tectum. This suggests that the LTTD-RC-tectal pathway of the rattlesnake may be homologous to the trigeminalreticular formation-tectal pathway of other species. On the other hand, Molenaar, in a FinkHeimer study of the python, describes a direct projection from LTTD to the contralateral tectum. The conflict between Molenaar's finding and our own work on the rattlesnake, where we find no such direct projection, could be due to species differences. The python and rattlesnake belong to two distantly related families of snakes. Thus in the python, the cells of RC, instead of forming a separate nucleus, may be intermingled with the cells of the LTTD. This is in keeping with the observations of Molenaar and Fizaan-Oostveen ('78) who, in a Fink-Heimer study, described an elaborate intranuclear projection within the LTTD. Auen ('78) recently reported that HRP injections in the tectum of the rattlesnake lead to retrograde filling of cells in the contralateral LTTD (called "LTD' by Auen). We cannot account for the difference between our work and his. We consistently filled cells of RC and not LTTD. We are puzzled by his results, because the location of his HRP-stained cells in the LTD are shown to be at the level of the VIII nerve root (his The tectal afferent groups we have found agree significantly with those found by Wilczynski and Northcutt ('77) in the frog. Following HRP tectal injections, they found that cell groups in the pretectal area of the lateral nucleus stained bilaterally, and that cells in the dorsal posterior nucleus and large-celled pretectal nucleus stained ipsilaterally. In the tegmentum the anterodorsal, posterodorsal and posteroventral fields had HRP-stained cells ipsilaterally; the ventral preoptic nucleus and suprapeduncular nucleus were stained bilaterally, and the nucleus isthmi was stained ipsilaterally. In the frog, Gruberg and Udin ('78) found bilateral input to the tectum from the nucleus isthmi. Ulinski ('77) used degeneration staining to study tecta efferent connections in the banded water snake. He found bilateral projections to the brainstem reticular formation, the nucleus lentiformis mesencephali, lateral habenular nuclei and posterodorsal nuclei and the dorsal lateral geniculate nucleus (densely). He found ipsilateral projections to the ventral lateral geniculate, the suprapeduncular nucleus, the caudal thalamus (diffusely throughout its central part) and the basal optic nucleus. In addition he found fibers in the contralateral tectum. There is thus substantial similarity between well-known connections of cold-blooded vertebrate tecta and connections that we have demonstrated in rattlesnake. In addition, we have found a nucleus in the medulla, the RC, which is a strong candidate for the link that relays infrared information from the LTTD to the intermediate layers of the optic tectum. Possible parallels of this trigemino-tectal pathway exist in mammals. It now remains to be determined whether the RC in rattlesnakes contains infrared-sensitive neurons and whether its mammalian counterpart, if it exists, is involved in thermoreception. ACKNOWLEDGMENT

Effect of long-term vitamin E and selenium supplementation on urine F2-isoprostanes, a biomarker of oxidative stress

BackgroundCigarette smoking generates reactive oxidant species and contributes to systemic oxidative stress, which plays a role in the pathophysiology of chronic diseases. Nutrients with antioxidant properties, including vitamin E and selenium, are proposed to reduce systemic oxidative burden and thus to mitigate the negative health effects of reactive oxidant species.ObjectiveOur objective was to determine whether long-term supplementation with vitamin E and/or selenium reduces oxidative stress in smokers, as measured by urine 8-iso-prostaglandin F2-alpha (8-iso-PGF2α).DesignWe measured urine 8-iso-PGF2α with competitive enzyme linked immunoassay (ELISA) in 312 male current smokers after 36 months of intervention in a randomized placebo-controlled trial of vitamin E (400IU/d all rac-α-tocopheryl acetate) and/or selenium (200µg/d L-selenomethionine). We used linear regression to estimate the effect of intervention on urine 8-iso-PGF2α, with adjustments for age and race.ResultsCompared to placebo, vitamin E alone lowered urine 8-iso-PGF2α by 21% (p=0.02); there was no effect of combined vitamin E and selenium (intervention arm lower by 9%; p=0.37) or selenium alone (intervention arm higher by 8%; p=0.52).ConclusionsLong-term vitamin E supplementation decreases urine 8-iso-PGF2α among male cigarette smokers, but we observed little to no evidence for an effect of selenium supplementation, alone or combined with vitamin E

Moving a randomized clinical trial into an observational cohort.

The Selenium and Vitamin E Cancer Prevention Trial (SELECT) was a randomized, double-blind, placebo-controlled prostate cancer prevention study funded by the National Cancer Institute (NCI) and conducted by the Southwest Oncology Group (SWOG). A total of 35,533 men were assigned randomly to one of the four treatment groups (vitamin E + placebo, selenium + placebo, vitamin E + selenium, and placebo + placebo). The independent Data and Safety Monitoring Committee (DSMC) recommended the discontinuation of study supplements because of the lack of efficacy for risk reduction and because futility analyses demonstrated no possibility of benefit of the supplements to the anticipated degree (25% reduction in prostate cancer incidence) with additional follow-up. Study leadership agreed that the randomized trial should be terminated but believed that the cohort should be maintained and followed as the additional follow-up would contribute important information to the understanding of the biologic consequences of the intervention. Since the participants no longer needed to be seen in person to assess acute toxicities or to be given study supplements, it was determined that the most efficient and cost-effective way to follow them was via a central coordinated effort.A number of changes were necessary at the local Study Sites and SELECT Statistical Center to transition to following participants via a Central Coordinating Center. We describe the transition process from a randomized clinical trial to the observational Centralized Follow-Up (CFU) study.The process of transitioning SELECT, implemented at more than 400 Study Sites across the United States, Canada, and Puerto Rico, entailed many critical decisions and actions including updates to online documents such as the SELECT Workbench and Study Manual, a protocol amendment, reorganization of the Statistical Center, creation of a Transition Committee, development of materials for SELECT Study Sites, development of procedures to close Study Sites, and revision of data collection procedures and the process by which to contact participants.At the time of the publication of the primary SELECT results in December 2008, there were 32,569 men alive and currently active in the trial. As of 31 December 2011, 17,761 participants had been registered to the CFU study. This number is less than had been anticipated due to unforeseen difficulties with local Study Site institutional review boards (IRBs). However, from this cohort, we estimate that an additional 580 prostate cancer cases and 215 Gleason 7 or higher grade cancers will be identified. Over 109,000 individual items have been mailed to participants. Active SELECT ancillary studies have continued. The substantial SELECT biorepository is available to researchers; requests to use the specimens are reviewed for feasibility and scientific merit. As of April 2012, 12 proposals had been approved.The accrual goal of the follow-up study was not met, limiting our power to address the study objectives satisfactorily. The CFU study is also dependent on a number of factors including continued funding, continued interest of investigators in the biorepository, and the continued contribution of the participants. Our experience may be less pertinent to investigators who wish to follow participants in a treatment trial or participants in prevention trials in other medical areas.Extended follow-up of participants in prevention research is important to study the long-term effects of the interventions, such as those used in SELECT. The approach taken by SELECT investigators was to continue to follow participants centrally via an annual questionnaire and with a web-based option. The participants enrolled in the CFU study represent a large, well-characterized, generally healthy cohort. The CFU has enabled us to collect additional prostate and other cancer endpoints and longer follow-up on the almost 18,000 participants enrolled. The utility of the extensive biorepository that was developed during the course of the SELECT is enhanced by longer follow-up