Circulation of marker substances in the cerebrospinal fluid of an amphibian,Rana pipiens

Summary Solutions of fluorescein-labelled dextran or Evans blue-albumin were infused into the lateral cerebral ventricle of Rana pipiens. The subsequent distribution in the cerebrospinal fluid (CSF) was investigated between 2 and 24 h after infusion by freezing and examination of the cut blocks of the head and vertebral column of the stage of a freezing microtome. These marker substances move out of the ventricles into the subarachnoid space at the caudal end of the fourth ventricle and spread rapidly along the subarachnoid space of the spinal cord. The spreading of marker substances is slower into the brain subarachnoid space. When the marker is infused into the subarachnoid space of the forebrain, it becomes distributed throughout the subarachnoid space of the brain and spinal cord but not in the ventricles.Partial clearance of markers from the ventricles takes place within 5 h and total clearance within 8 h. Clearance from the brain and cord subarachnoid space is somewhat slower and can only be detected in experiments lasting 10 h or more. Absorption of the markers from the CSF occurs via the intervertebral foramina of the spinal cord. Fluorescence microscopy of sections of the cord show that the fluorescence leaves the subarachnoid space at the point where the spinal nerves traverse the arachnoid membrane.     

Preclinical pharmacokinetics of MHAA4549A,a human monoclonal antibody to influenza A virus,and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza A

MHAA4549A is a human immunoglobulin G1 (IgG1) monoclonal antibody that binds to a highly conserved epitope on the stalk of influenza A hemagglutinin and blocks the hemagglutinin-mediated membrane fusion in the endosome, neutralizing all known human influenza A strains. Pharmacokinetics (PK) of MHAA4549A and its related antibodies were determined in DBA/2J and Balb-c mice at 5 mg/kg and in cynomolgus monkeys at 5 and 100 mg/kg as a single intravenous dose. Serum samples were analyzed for antibody concentrations using an ELISA and the PK was evaluated using WinNonlin software. Human PK profiles were projected based on the PK in monkeys using species-invariant time method. The human efficacious dose projection was based on in vivo nonclinical pharmacological active doses, exposure in mouse infection models and expected human PK. The PK profiles of MHAA4549A and its related antibody showed a linear bi-exponential disposition in mice and cynomolgus monkeys. In mice, clearance and half-life ranged from 5.77 to 9.98 mL/day/kg and 10.2 to 5.76 days, respectively. In cynomolgus monkeys, clearance and half-life ranged from 4.33 to 4.34 mL/day/kg and 11.3 to 11.9 days, respectively. The predicted clearance in humans was ～2.60 mL/day/kg. A single intravenous dose ranging from 15 to 45 mg/kg was predicted to achieve efficacious exposure in humans. In conclusion, the PK of MHAA4549A was as expected for a human IgG1 monoclonal antibody that lacks known endogenous host targets. The predicted clearance and projected efficacious doses in humans for MHAA4549A have been verified in a Phase 1 study and Phase 2a study, respectively.     

A METHOD FOR THE STUDY OF CHOLESTEROL BIOSYNTHESIS IN THE CENTRAL NERVOUS SYSTEM

Abstract—

• 1 After intraperitoneal injection, there is negligible incorporation of [2-¹⁴C]-mevalonic lactone into the CNS of the adult rat.
• 2 Mevalonic lactone injected into the CSF is quickly transferred to blood.
• 3 Mevalonic lactone injected in the cistema magna or the lateral ventricle of the brain does not diffuse readily into the whole CSF. Spinal cord cholesterol is most heavily labelled after intracisternal injection, as is brain cholesterol after intraventricular administration.
• 4 After intraventricular perfusion, the diffusion of mevalonic lactone into the ventricle opposite the side of the injection is increased when the rate of perfusion is doubled from 5 to 10 μ1/hr. After injection, optimal homogeneity is obtained if a large volume (70μl) is administered.
• 5 An increase in the volume of injection from 70 μl to 130μl does not alter the distribution of activity between the left and right ventricles, nor does it increase the diffusion of mevalonic lactone from ventricle to spinal cord CSF.
• 6 The mean yield of mevalonic lactone incorporation into brain cholesterol is much higher after injection than after perfusion of precursor into the lateral cerebral ventricle.

     

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.     

Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge

Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development.     

Endogenous Content and Release of [3H]-GABA and [3H]-Glutamate in the Spinal Cord of Chronically Undernourished Rat

The aim of this study was to determine the effect of chronic undernutrition on the content and release of γ-amino butyric acid (GABA) and glutamate (GLU) transmitters in the rat spinal cord. The release of [³H]-GABA and [³H]-GLU was determined by radioactive liquid scintillation techniques, and the concentrations of GABA and GLU in spinal cord preparations from control and undernourished young rats (50–60 days old) were measured by reverse-phase HPLC. The GABA and GLU contents in the lumbar spinal dorsal horn (L6 segment) were significantly lower in undernourished rats relative to control rats (22.2 ± 3.7 and 10.7 ± 1.9 %, respectively; P < 0.05). Spinal cord blocks from undernourished animals also showed lower rates of [³H]-GABA and [³H]-GLU release than controls (27.6 ± 3.5 and 12.8 ± 2.5 %, respectively; P < 0.01). We propose that the decreases in GLU content and release are consistent with a reduced activation of either afferent fibers, spinal glutaminergic neurons, or both. Furthermore, we propose that the decreased content and release of GABA in undernourished animals are related to a depression in pre- and post-synaptic inhibition. In addition, we hypothesize that the reductions in GABA content and release serve as compensatory mechanisms to counterbalance decreases in sensory transmission and GLU content in the spinal cord of the chronically undernourished rat.     

Stimulating Effects of Sucrose and Inulin on Growth,Lactate, and Bacteriocin Productions by <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis>

Sucrose and inulin, when combined with glucose, behaved as stimulating agents of bacteriocin production by Pediococcus pentosaceus ATCC 43200. When such microbial strain was grown in glucose-based Man, Rogosa, and Sharpe (MRS) medium, without any additional supplement, it showed higher maximum cell concentration (2.68 ± 1.10 g/L) and longer generation time (2.17 ± 0.02 h), but lower specific growth rate (0.32 ± 0.01 h^?1) than in the same medium supplemented with 1.0% of both ingredients (2.53 ± 1.10 g/L, 1.60 ± 0.05 h and 0.43 ± 0.02 h^?1, respectively). Glucose replacement by sucrose or inulin almost completely suppressed growth, hence confirming that it is the preferred carbon source for this strain. Qualitatively, similar results were observed for lactate production, which was 59.8% higher in glucose-based medium. Enterococcus and Listeria strains were sensitive to bacteriocin, whose antimicrobial effect after 8 h increased from 120.25 ± 0.35 to 144.00 ± 1.41 or 171.00 ± 1.41 AU/mL when sucrose or inulin was added to the glucose-based MRS medium. Sucrose and inulin were also able to speed up P. pentosaceus growth in the exponential phase.     

Numerical study of intrathecal drug delivery to a permeable spinal cord: effect of catheter position and angle

Intrathecal delivery is a procedure involving the release of therapeutic agents into the cerebrospinal fluid (CSF) hrough a catheter. It holds promise for treating high-impact central nervous system pathologies, for which systemic administration routes are ineffective. In this study we introduce a numerical model able to simultaneously account for solute transport in the fluid and in the spinal cord. Using a Discontinuous Galerkin method and a three-dimensional patient-specific geometry, we studied the effect of catheter position and angle on local spinal cord drug concentration. We considered twenty cardiac cycles to limit the computational cost of our approach, which resolves the physics both in space and time. We used clinically representative data for the drug injection speed and dose rate, and scaled drug diffusion/penetration properties to obtain observable effects during the considered simulation time. Based on our limited set of working parameters, lateral injection perpendicular to the cord turned out to be more effective than other configurations. Even if the adopted scaling does not allow for a direct clinical translation (a wider parametric assessment of the importance of CSF flow, geometry and diffusion properties is needed), it did not weaken our numerical approach, which can be used to systematically investigate multiple catheter, geometry and fluid/tissue properties configurations, thus paving the way for therapy control.     

Feeding in satiated sheep elicited by intraventricular injections of CSF from fasted sheep

Cerebrospinal fluid (CSF) was collected from the lateral ventricles of either 24-hr fasted or “satiated” sheep (donors) and injected into the lateral or third ventricle of either 24-hr fasted or “satiated” sheep (recipients). Recipient “satiated” sheep ate more following injections of CSF from fasted donors, as compared to when the same animals were injected with CSF from equally “satiated” donors (e.g., 73±10 vs 16±6g respectively, 15 min post-injection, P <0.01). Feed intake of fasted recipient sheep was slightly depressed following intraventricular injections of CSF from “satiated” donors. Apparently, the composition of cerebrospinal fluid of the donor sheep was affected by the surfeit-deficit state of their energy stores.     

Preclinical pharmacokinetics and pharmacodynamics of DCLL9718A: An antibody-drug conjugate for the treatment of acute myeloid leukemia

ABSTRACT

Few treatment options are available for acute myeloid leukemia (AML) patients. DCLL9718A is an antibody-drug conjugate that targets C-type lectin-like molecule-1 (CLL-1). This receptor is prevalent on monocytes, neutrophils, and AML blast cells, and unlike CD33, is not expressed on hematopoietic stem cells, thus providing possible hematopoietic recovery. DCLL9718A comprises an anti-CLL-1 IgG1 antibody (MCLL0517A) linked to a pyrrolobenzodiazepine (PBD) dimer payload, via a cleavable disulfide-labile linker. Here, we characterize the in vitro and in vivo stability, the pharmacokinetics (PK) and pharmacodynamics (PD) of DCLL9718A and MCLL0517A in rodents and cynomolgus monkeys. Three key PK analytes were measured in these studies: total antibody, antibody-conjugated PBD dimer and unconjugated PBD dimer. In vitro, DCLL9718A, was stable with most (> 80%) of the PBD dimer payload remaining conjugated to the antibody over 96 hours. This was recapitulated in vivo with antibody-conjugated PBD dimer clearance estimates similar to DCLL9718A total antibody clearance. Both DCLL9718A and MCLL0517A showed linear PK in the non-binding rodent species, and non-linear PK in cynomolgus monkeys, a binding species. The PK data indicated minimal impact of conjugation on the disposition of DCLL9718A total antibody. Finally, in cynomolgus monkey, MCLL0517A showed target engagement at all doses tested (0.5 and 20 mg/kg) as measured by receptor occupancy, and DCLL9718A (at doses of 0.05, 0.1 and 0.2 mg/kg) showed strong PD activity as evidenced by notable reduction in monocytes and neutrophils.     

Cerebrospinal fluid-contacting neurons of the central canal and terminal ventricle in various vertebrates

Cell and Tissue Research - Cerebrospinal fluid (CSF)-contacting neurons were studied by means of electron microscopy in the spinal cord and/or terminal ventricle of the ray, Raja clavata...     

Pressure-dependent PAH and creatinine transport from the brain ventricles.

The brain ventricular system of the adult dog was perfused with an artificial cerebrospinal fluid (CSF) containing inulin, creatinine and radioactively labeled p-aminohippuric acid (PAH) and mannitol. Inflow and outflow rates and concentrations of test molecules were measured at different intraventricular pressures, allowing calculation of their steady-state rates of removal from the ventricles. Clearance of inulin, a measure of CSF bulk absorption varied nearly with intraventricular pressure (- 15 to +12 cm H2O relative to the external auditory meatus). The efflux coefficient (Ko; representing clearance of a molecule by means other than bulk absorption) for mannitol was independent of intraventricular pressure. Ko's for PAH and creatinine were pressure dependent. PAH and creatinine efflux may be related to the amount of fourth ventricular choroid plexus surface exposed to the perfusion fluid. Ko's for creatinine and PAH (46 plus or minus 4 mul/min; 34 plus or minus 4mul/min, respectively) were significantly greater than mannitol (16 plus or minus 8 mul/min) at comparable intraventricular pressures, suggesting that both creatinine and PAH leave the CSF by an active process in addition to passive diffusion.     

Comparative effect of different dietary inulin sources and probiotics on growth performance and blood characteristics in growing–finishing pigs

The study was focused on assessment of the effect of an extract of long-chain inulin (LCI) and dried tubers of Jerusalem artichoke (JA) and a multispecies probiotic preparation as well as a combination thereof on growth performance and blood parameters of fattening pigs. In total, 144 pigs (initial body weight 30.0 ± 0.5 kg) were used in a 98-d experiment. The six dietary treatments consisted of the control diet (Con), diet Con supplemented with probiotics (ConP) and four diets supplemented with LCI or JA alone or with probiotics (diets LCIP and JAP). Throughout the fattening period, there was a beneficial effect of the probiotic supplementation to the inulin-containing diets and the average daily gain (ADG) was increased by supplementation of probiotics in combination with inulin sources (p < 0.05). At the end of the fattening period, ADG and feed conversion ratio (FCR) were higher after supplementation of LCI only (p < 0.05). Compared with group ConP, in groups LCI and JA, the ADG and FCR were improved (p < 0.05). Only in the first fattening stage, the addition of the prebiotics and/or probiotics had an impact on the level of white blood cells and some biochemical indices in pigs. In younger animals, probiotic or LCI supplementation increased the IgG level (p < 0.05). There was also an interaction between the probiotics and JA resulting in increased IgG and IgA concentrations (p < 0.05). In the finishing period, LCI addition increased the IgM level (p < 0.05), whereas JA addition increased IgG and IgM levels as well (p < 0.05). In conclusion, both dietary sources of inulin and probiotic supplementation can improve the fattening performance and health status of growing pigs.     

Cerebrospinal fluid contacting neurons in the reduced brain ventricular system of the atlantic hagfish,Myxine glutinosa

Cerebrospinal fluid (CSF)-contacting neurons are sensory-type cells sending ciliated dendritic process into the CSF. Some of the prosencephalic CSF-contacting neurons of higher vertebrates were postulated to be chemoreceptors detecting the chemical composition of the CSF, other cells may percieve light as "deep encephalic photoreceptors". In our earlier works, CSF-contacting neurons of the mechanoreceptor-type were described around the central canal of the hagfish spinal cord. It was supposed that perceiving the flow of the CSF they are involved in vasoregulatory mechanisms of the nervous tissue. In the present work, we examined the brain ventricular system of the Atlantic hagfish with special reference to the presence and fine structure of CSF-contacting neurons. Myxinoids have an ontogenetically reduced brain ventricular system. In the adult hagfish (Myxine glutinosa) the lumen of the lateral ventricle is closed, the third ventricle has a preoptic-, infundibular and subhabenular part that are not connected to each other. The choroid plexus is absent. The infundibular part of the third ventricle has a medial hypophyseal recess and, more caudally, a paired lateral recess. We found CSF-contacting neurons in the lower part of the third ventricle, in the preoptic and infundibular recess as well as in the lateral infundibular recesses. No CSF-contacting neurons were found in the cerebral aqueduct connecting the subhabenular recess to the fourth ventricle. There is a pineal recess and a well-developed subcommissural organ at the rostral end of the aqueduct. Extending from the caudal part of the fourth ventricle in the medulla to the caudal end of the spinal cord, the central canal has a dorsal and ventral part. Dendrites of CSF-contacting neurons are protruding into the ventral lumen. Corroborating the supposed choroid plexus-like function of the wall of the dorsal central canal, segmental vessels reach a thin area on both sides of the ependymal lining. The perikarya of the CSF-contacting neurons found in the brain ventricles are mainly bipolar and contain granular vesicles of various size. The bulb-like terminal of their ventricular dendrites bears several stereocilia and contains basal bodies as well as mitochondria. Basal bodies emit cilia of the 9+0-type. Cilia may arise from the basal body and accessory basal body as well. The axons run ependymofugally and enter--partially cross--the periventricular synaptic zones. No neurohemal terminals similar to those formed by spinal CSF-contacting neurons of higher vertebrates have been found in the hagfish. We suppose that CSF-contacting neurons transform CSF-mediated non-synaptic information taken up by their ventricular dendrites to synaptic one. A light-sensitive role for some (preoptic?) groups of CSF-contacting neurons cannot be excluded.     

Integrated pharmacokinetic,pharmacodynamic and immunogenicity profiling of an anti-CCL21 monoclonal antibody in cynomolgus monkeys

QBP359 is an IgG1 human monoclonal antibody that binds with high affinity to human CCL21, a chemokine hypothesized to play a role in inflammatory disease conditions through activation of resident CCR7-expressing fibroblasts/myofibroblasts. The pharmacokinetics (PK) and pharmacodynamics (PD) of QBP359 in non-human primates were characterized through an integrated approach, combining PK, PD, immunogenicity, immunohistochemistry (IHC) and tissue profiling data from single- and multiple-dose experiments in cynomolgus monkeys. When compared with regular immunoglobulin typical kinetics, faster drug clearance was observed in serum following intravenous administration of 10 mg/kg and 50 mg/kg of QBP359. We have shown by means of PK/PD modeling that clearance of mAb-ligand complex is the most likely explanation for the rapid clearance of QBP359 in cynomolgus monkey. IHC and liquid chromatography mass spectrometry data suggested a high turnover and synthesis rate of CCL21 in tissues. Although lymphoid tissue was expected to accumulate drug due to the high levels of CCL21 present, bioavailability following subcutaneous administration in monkeys was 52%. In human disease states, where CCL21 expression is believed to be expressed at 10-fold higher concentrations compared with cynomolgus monkeys, the PK/PD model of QBP359 and its binding to CCL21 suggested that very large doses requiring frequent administration of mAb would be required to maintain suppression of CCL21 in the clinical setting. This highlights the difficulty in targeting soluble proteins with high synthesis rates.     

Establishment of a Spinal Cord Injury Model in Adult Rats by an Electrocircuit-Controlled Impacting Device and its Pathological Observations

One of the crucial challenges in medicine is the treatment and rehabilitation of spinal cord injury (SCI). In this study, we established a stable and reproducible acute spinal cord injury model in adult rats. The SCI was inflicted by our self-innovated spinal cord impact device controlled by electrical circuit. The Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB) score, electrophysiology, histological, and immunohistochemical changes after SCI were observed. The BBB score of the injured rats began to increase from the 3rd day of SCI and reached at the score 7.2 ± 1.3 at the 28th day. The latency of cortical somatosensory evoked potentials (CSEP) was not observed 2 and 6 h after injury, but appeared 24 h after injury which was significantly prolonged. It recovered from day 3 gradually to 27.3 ± 2.7 ms on day 28. H&E staining showed that the structure of gray and white matter was disrupted after the SCI. The result also showed dramatic neuron degenerations, cellular swelling, and the proliferation of glial cells. The immunohistochemical analysis showed that the expression of neuron specific enolase (NSE) and neurofilament 200 (NF200) started lowering at 2 h and dropped to the bottom at 24 h. Their expression rebound from day 3 and yet to the original level at day 28 (P < 0.05). The number of cells expressing glial fibrillary acidic protein (GFAP) hiked from day 3, peaked at day 14, and began recovering from day 28 (P < 0.05). The changes of NSE, NF200, GFAP, and CSEP were significantly associated with the BBB score (P < 0.05). In conclusion, our self-innovated device can reproduce the injury model stably. The changes of NSE, NF, and GFAP after spinal cord injury reflect the characteristics of pathological change, which are closely associated with the functional recovery from the spinal cord injury.     

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.     

Method for cisterna magna perfusion of synthetic cerebrospinal fluid in the awake goat

We developed a method to produce stable alterations in the ionic composition of the medullary chemoreceptor environment. A double-lumen catheter system (Hustead epidural needle and epidural catheter) was placed through a plastic cisternal guide tube into the cisterna magna of awake goats. A push-pull perfusion system using a modified infusion pump delivered matched cerebrospinal fluid (CSF) perfusate inflow and outflow of 3.1 ml/min. Ventilation changed within 15 min of the initiation of perfusion and reached steady state within 45-65 min. Steady-state ventilatory responses could be maintained for up to 240 min and were readily reversed in response to a change in [HCO-3]. Perfusions with normal mock CSF ( [HCO-3] = 23 meq/l) caused no change from nonperfused values. Over the range of CSF perfusate [HCO-3] used (13.5-34.4 meq/l), the gain of the steady-state ventilatory response averaged 0.6 Torr X meq-1 X l. [3H]inulin and [HCO-3] were equal in inflow and outflow by 20-30 min of perfusion indicating complete mixing of bulk CSF in the cistern. Anatomic study after methylene blue dye perfusion showed dye distribution to subarachnoid spaces of midbrain, cervical cord, cerebellum, medulla, and most of the cortex but not to any ventricles. This perfusion technique produces prolonged, stable, reproducible, and repeatable changes in the medullary chemoreceptor ionic environment of awake goats, is relatively atraumatic, and permits high flow through the cisternal subarachnoid space.     

Immunoglobulins and complement in demyelination induced in mice by Theiler's virus

Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) produces chronic demyelination and persistent infection in the central nervous system (CNS) of susceptible SJL mice. This series of experiments examined the contribution of humoral immunity and C to myelin destruction. As in multiple sclerosis, mice persistently infected with TMEV had elevated levels of IgG and oligoclonal bands in the cerebrospinal fluid (CSF). Immunoblot studies revealed that even in animals exhibiting profound demyelination, IgG in the serum and CSF was directed primarily at virus antigen rather than at normal myelin components. Inflammatory cells positive for Ig were distributed mainly around blood vessels, but occasionally they infiltrated the spinal cord parenchyma. Rare examples of myelin sheaths positive for IgG were found by immunoelectron microscopy in spinal cord sections from infected mice; the third component of complement (C3) was commonly found in the walls of CNS blood vessels but not on myelin. Neither serum nor CSF IgG from infected mice bound to myelin sheaths or other CNS components in sections of normal syngeneic spinal cord. There were significantly more demyelinating lesions in infected mice depleted of C components with cobra venom factor. These data do not support a humoral autoimmune basis for the CNS demyelination that occurs in association with persistent TMEV infection. However, the humoral immune response directed at TMEV antigens may either limit virus spread or promote virus persistence.