A rhesus monkey model for continuous infusion of drugs into cerebrospinal fluid

A new rhesus monkey model with two intraventricular catheter systems was developed to examine the pharmacokinetics and neurotoxicity of chemotherapeutic agents administered by continuous intraventricular infusion. A lateral ventricular catheter system implanted in the lateral ventricle and attached to a subcutaneous access port on the animal's back is used for infusion of drugs into the ventricle. A Pudenz catheter implanted in the fourth ventricle and connected to a subcutaneous Ommaya reservoir permits repetitive CSF sampling in unanesthetized animals. The model was evaluated in five animals for over 12 months for catheter patency, surgical complications, and utility in studying the pharmacokinetics of continuous intraventricular infusion of methotrexate. There were no perioperative complications. Three of the five monkeys maintained both systems successfully. The other two animals developed staphylococcal ventriculitis, one at 7 days as a result of manipulation of the incision by the animal leading to cellulitis around the catheter site and subsequent ventriculitis, the other at 5 months. Both animals were treated successfully with antibiotics and catheter removal. An infusion of 0.05 mg of methotrexate over 24 hours maintained ventricular drug concentrations of 1 mol/L without evidence of neurotoxicity. This new model has applications both for the development of continuous intraventricular infusion as a therapeutic approach for the treatment of meningeal cancers in humans and as a research tool to study the distribution and elimination of drugs from the CSF.     

Development of a Cerebrospinal Fluid Lateral Reservoir Model in Rhesus Monkeys (Macaca mulatta)

Rapid, serial, and humane collection of cerebrospinal fluid (CSF) in nonhuman primates (NHP) is an essential element of numerous research studies and is currently accomplished via two different models. The CSF reservoir model (FR) combines a catheter in the 4th ventricle with a flexible silastic reservoir to permit circulating CSF flow. The CSF lateral port model (LP) consists of a lateral ventricular catheter and an IV port that provides static access to CSF and volume restrictions on sample collection. The FR model is associated with an intensive, prolonged recovery and frequent postsurgical hydrocephalus and nonpatency, whereas the LP model is associated with an easier recovery. To maximize the advantages of both systems, we developed the CSF lateral reservoir model (LR), which combines the beneficial features of the 2 previous models but avoids their limitations by using a reservoir for circulating CSF flow combined with catheter placement in the lateral ventricle. Nine adult male rhesus monkeys were utilized in this study. Pre-surgical MRI was performed to determine the coordinates of the lateral ventricle and location of choroid plexus (CP). The coordinates were determined to avoid the CP and major blood vessels. The predetermined coordinates were 100% accurate, according to MRI validation. The LR system functioned successfully in 67% of cases for 221 d, and 44% remain functional at 426 to 510 d postoperatively. Compared with established models, our LR model markedly reduced postoperative complications and recovery time. Development of the LR model was successful in rhesus macaques and is a useful alternative to the FR and LP methods of CSF collection from nonhuman primates.Abbreviations: CP, choroid plexus; FR, CSF 4th ventricular reservoir model; LP, CSF lateral port model; LR, CSF lateral reservoir model; SER, successful establishment rateSerial ventricular CSF sampling in NHP is a frequent and critical requirement for a wide variety of studies and is predominantly accomplished by using either of 2 models. The 4th ventricle (FR) model, previously referred to as an Ommaya reservoir,⁶ and lateral port (LP) models² are closed, indwelling, subcutaneous systems that allow for serial, rapid, and humane collection of CSF, as well as intraventricular drug administration, in unanesthetized and restrained NHP.The FR model (Figure 1 A) consists of a catheter that is placed in the 4th ventricle and attached to a silastic reservoir that is implanted subcutaneously over the occipital bone. The silastic reservoir is depressed repetitively prior to and after sampling to circulate the CSF throughout the ventricles and catheter system to provide an unbiased sample without volume loss to dead space. The reservoir is accessed percutaneously to obtain a CSF sample via aspiration or to administer drug. The FR model initially was developed in 1977⁶ and continues to be used for pharmacokinetic studies. This system continues to demonstrate a low rate of successful establishment, but once established, the FR model remains patent for prolonged periods without evidence of neurologic sequelae or bleeding from the choroid plexus (CP) in rhesus macaques. The decreased establishment rate of this model is attributed, at least in part, to postsurgical development of hydrocephalus, given that the catheter, which is routed through the aqueduct of Magendie to the 4th ventricle, can obstruct the flow of CSF. In addition, maintaining catheter patency is problematic due to CP bleeding during the recovery period. Postsurgical care and recovery after creating the FR are extensive, frequently requiring prolonged analgesics and steroid administration, with many days needed for complete recovery.Open in a separate windowFigure 1.Evolution of CSF ventricular models, with flow dynamics. (A) Sagittal diagram of original FR (Ommaya) model, with placement in the 4th ventricle. Developed in 1977. Arrows indicate the circulating flow of CSF. (B) Original LP model, with catheter placement in the lateral ventricle and attachment to an IV access port. Developed in 1990. Arrows indicate the static, unidirectional flow of CSF. (C) The LR model, a composite of the 2 earlier CSF models. Arrows indicate the circulating flow of CSF.The LP model (Figure 1 B) consists of a catheter that is implanted in the lateral ventricle and attached to a subcutaneous intravenous access port. The port is accessed percutaneously to obtain the CSF sample or to administer a drug. The LP is a static model, because the CSF is not circulated or mixed through the ventricles, and CSF is obtained via unidirectional flow. The LP model was developed in 1990 for intrathecal drug administration³ and has been used subsequently for CSF collection⁴ by several investigators. CSF sampling with the LP model is restrictive: the volume of the system (that is, the dead space) must be removed at each collection to obtain an unbiased sample, collection is accomplished via gravitational flow and not aspiration, and the collection frequency is dependent on the rate of CSF replacement. In addition, the potential for sample contamination from blood due to CP bleeding remains problematic for the duration of LP implantation. However, the use of the lateral ventricle avoids the postsurgical complication of hydrocephalus. This system demonstrated a high rate of successful establishment with a reduction in the necessary analgesic and steroid administration as well as days to complete recovery, as compared with the FR model. Analysis of our clinical records from 2003 to 2013 revealed a successful establishment rate (SER) of 39% for the FR model; 33% of these systems remained functional for 3 to 7.5 y (

An overview of the biology and status of undulate ray Raja undulata in the north-east Atlantic Ocean

The undulate ray Raja undulata is one of the lesser-known skates occurring on the continental shelf of the north-east Atlantic Ocean. It is patchily distributed throughout its range, with sites of local abundance in the central English Channel and off the coasts of Ireland, France, Spain and Portugal. Raja undulata is most abundant in coastal waters (<50 m deep) and is often found in proximity to large estuaries, rias and bays. It is a relatively large-bodied species, attaining a maximum total length (L(T) ) of at least 114 cm, with females maturing at an L(T) of c. 84 cm in Portuguese waters. Although infrequently taken in existing trawl surveys, it can be locally abundant in certain areas, where it can be the dominant skate species. Given its large size, patchy distribution and concern over the possibilities of localized depletions, the IUCN listed R. undulata as an endangered species and, since 2009, the European Union has established regulations to prohibit commercial fisheries landing the species. Given the increased interest in the species, a synopsis of current knowledge is provided, and available data from internationally co-ordinated trawl surveys presented.     

A new Sphagesaurus (Mesoeucrocodylia: Notosuchia) from the Upper Cretaceous of Monte Alto City (Bauru Group,Brazil), and a revision of the Sphagesauridae

Oxygen consumption and ammonia excretion rates were assessed for Terebratulina retusa (L.) held under 3 different regimes of temperature and food availability. These were: 5.6?C, no food (cold, starved); 5.8?C, food present (cold, fed) and 10.7?C food present (warm, fed), which simulated winter conditions, summer conditions and an intermediate treatment. Regressions of oxygen consumption on ash‐free dry weight (AFDW) had slopes which were not significantly different from each other and ranged from 0.953 to 0.999. A common slope of 0.976 was calculated and intercepts based on the common slope used to compare oxygen consumption in each treatment. The rise from cold, starved conditions to warm, fed was 24.5 per cent and this was significant (P < 0.05). Other differences were not significant (P > 0.05) but the cold, fed result was 12.6 per cent higher than cold, starved. Therefore feeding and temperature probably account for equivalent proportions of the rise in metabolism from winter to summer. Ammonia production data were much more variable. Excretion rates of a 50 mg AFDW individual (in ng‐at NH₃‐N.h^‐1) were as follows: cold, starved: 30.2 cold, fed: 7.1; and warm, fed: 22.9. Oxygen to nitrogen (O:N) ratios reflected these results. Mean O:N ratios were: cold, starved: 8.0; cold, fed: 42.4; warm, fed: 16.3. This shows that the simulated winter group relied heavily on protein to fuel their metabolism, the simulated summer group were less dependent on protein and the intermediate group probably used lipids and carbohydrates to fuel metabolic demands. This possibly reflected a trade off between food supply and increased metabolism from treatment to treatment, demonstrating a flexibility which could have been a contributing factor in the ecological tolerance and geological longevity of some brachiopods.     

Noninvasive evaluation of skeletal muscle mitochondrial capacity with near-infrared spectroscopy: correcting for blood volume changes

Near-infrared spectroscopy (NIRS) is a well-known method used to measure muscle oxygenation and hemodynamics in vivo. The application of arterial occlusions allows for the assessment of muscle oxygen consumption (mVo(2)) using NIRS. The aim of this study was to measure skeletal muscle mitochondrial capacity using blood volume-corrected NIRS signals that represent oxygenated hemoglobin/myoglobin (O(2)Hb) and deoxygenated hemoglobin/myoglobin (HHb). We also assessed the reliability and reproducibility of NIRS measurements of resting oxygen consumption and mitochondrial capacity. Twenty-four subjects, including four with chronic spinal cord injury, were tested using either the vastus lateralis or gastrocnemius muscles. Ten healthy, able-bodied subjects were tested on two occasions within a period of 7 days to assess the reliability and reproducibility. NIRS signals were corrected for blood volume changes using three different methods. Resting oxygen consumption had a mean coefficient of variation (CV) of 2.4% (range 1-32%). The recovery of oxygen consumption (mVo(2)) after electrical stimulation at 4 Hz was fit to an exponential curve, which represents mitochondrial capacity. The time constant for the recovery of mVo(2) was reproducible with a mean CV of 10% (range 1-22%) only when correcting for blood volume changes. We also examined the effects of adipose tissue thickness on measurements of mVo(2). We found the mVo(2) measurements using absolute units to be influenced by adipose tissue thickness (ATT), and this relationship was removed when an ischemic calibration was performed, supporting its use to compare mVo(2) between individuals of varying ATT. In conclusion, in vivo oxidative capacity can be assessed using blood volume-corrected NIRS signals with a high degree of reliability and reproducibility.     

Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO.

Branched-chain oxo acid dehydrogenase was purified from Pseudomonas aeruginosa strain PAO with the objective of resolving the complex into its subunits. The purified complex consisted of four proteins, of Mr 36,000, 42,000, 49,000 and 50,000. The complex was resolved by heat treatment into the 49,000 and 50,000-Mr proteins, which were separated by chromatography on DEAE-Sepharose. The 49,000-Mr protein was identified as the E2 subunit by its ability to catalyse transacylation with a variety of substrates, with dihydrolipoamide as the acceptor. P. aeruginosa, like P. putida, produces two lipoamide dehydrogenases. One, the 50,000-Mr protein, was identified as the specific E3 subunit of branched-chain oxo acid dehydrogenase and had many properties in common with the lipoamide dehydrogenase LPD-val of P. putida. The second lipoamide dehydrogenase had Mr 54,000 and corresponded to the lipoamide dehydrogenase LPD-glc of P. putida. Fragments of C-terminal CNBr peptides of LPD-val from P. putida and P. aeruginosa corresponded closely, with only two amino acid differences over 31 amino acids. A corresponding fragment at the C-terminal end of lipoamide dehydrogenase from Escherichia coli also showed extensive homology. All three peptides had a common segment of eight amino acids, with the sequence TIHAHPTL. This homology was not evident in any other flavoproteins in the Dayhoff data base which suggests that this sequence might be characteristic of lipoamide dehydrogenase.     

Increasing blood flow before exercise in spinal cord-injured individuals does not alter muscle fatigue.

Previous studies have shown increased fatigue in paralyzed muscle of spinal cord-injured (SCI) patients (Castro M, Apple D Jr, Hillegass E, and Dudley GA. Eur J Appl Physiol 80: 373-378, 1999; Gerrits H, Hopman MTE, Sargeant A, and de Haan A. Clin Physiol 21: 105-113, 2001). Our purpose was to determine whether the increased muscle fatigue could be due to a delayed rise in blood flow at the onset of exercise in SCI individuals. Isometric electrical stimulation was used to induce fatigue in the quadriceps femoris muscle of seven male, chronic (>1 yr postinjury), complete (American Spinal Injury Association, category A) SCI subjects. Cuff occlusion was used to elevate blood flow before electrical stimulation, and the magnitude of fatigue was compared with a control condition of electrical stimulation without prior cuff occlusion. Blood flow was measured in the femoral artery by Doppler ultrasound. Prior cuff occlusion increased blood flow in the first 30 s of stimulation compared with the No-Cuff condition (1,350 vs. 680 ml/min, respectively; P < 0.001), although blood flow at the end of stimulation was the same between conditions (1,260 +/- 140 vs. 1,160 +/- 370 ml/min, Cuff and No-Cuff condition, respectively; P = 0.511). Muscle fatigue was not significantly different between prior cuff occlusion and the control condition (32 +/- 13 vs. 35 +/- 10%; P = 0.670). In conclusion, increased muscle fatigue in SCI individuals is not associated with the prolonged time for blood flow to increase at the onset of exercise.     

Blood flow and muscle fatigue in SCI individuals during electrical stimulation.

Our purpose was to measure blood flow and muscle fatigue in chronic, complete, spinal cord-injured (SCI) and able-bodied (AB) individuals during electrical stimulation. Electrical stimulation of the quadriceps muscles was used to elicit similar activated muscle mass. Blood flow was measured in the femoral artery by Doppler ultrasound. Muscle fatigue was significantly greater (three- to eightfold, P < or = 0.001) in the SCI vs. the AB individuals. The magnitude of blood flow was not significantly different between groups. A prolonged half-time to peak blood flow at the beginning of exercise (fivefold, P = 0.001) and recovery of blood flow at the end of exercise (threefold, P = 0.009) was found in the SCI vs. the AB group. In conclusion, the magnitude of the muscle blood flow to electrical stimulation was not associated with increased muscle fatigue in SCI individuals. However, the prolonged time to peak blood flow may be an explanation for increased fatigue in SCI individuals.     

The rhizosphere in Zea: new insight into its structure and development

Some of the nodal roots of field-grown Zea mays L. bear a persistent soil sheath along their entire length underground except for a glistening white soil-free zone which extends approximately 25 mm behind the root cap. These roots are generally unbranched. The histology of the surface and the rhizosphere of the sheathed roots has been examined by correlated light and electron microscopy. All mature peripheral tissues including root hairs, are largely intact and apparently alive where enclosed by the soil sheath. The sheath is permeated by extracellular mucilage which is histochemically distinct from the mucilage at the epidermal surface, but similar to that produced by the root cap. Isolated cells resembling those sloughed from the sides of the root cap persist in the soil sheath along the length of these roots. Fresh whole mounts of the sheath show that these detached cells may be alive and streaming vigorously even at some distance from the root cap. Rhizosphere mucilage is associated with the isolated cells.To whom correspondence should be addressed