Andrew A. Monjan

Manipulation of mobile phase parameters for the HPLC separation of endogenous monoamines in rat brain tissue

Multi-component mobile phases are common in ion pair reverse phase high performance liquid chromatography for the analysis of endogenous monoamines in rat brain tissue. By examining the effects of each component of a mobile phase on the separation of a number of monoamines and their metabolites, we show how optimization of parameters can achieve and/or maintain a separation both within and between different octadecyl columns. The assay itself is rapid, sensitive, demands minimal sample preparation, and results in complete resolution of the amines. More importantly, the basis for the manipulation of mobile phase components is discussed with regard to practical utilization in selective amine separation. We conclude that knowledge of mobile phase parameters, their mechanism, and manipulation is as important as the assay itself.

High-Performance Liquid Chromatographic (HPLC) Separation and Quantitation of Endogenous Glucocorticoids after Solid-Phase Extraction from Plasma

This study describes a method for the extraction and simultaneous measurement of cortisone, cortisol and corticosterone using dexamethasone as an internal standard. Solid-phase extraction of plasma steroids with C18 columns allows the samples to be extracted, washed and concentrated in a single step with minimal sample handling and without the use of large volumes of organic solvents. HPLC separation of the steroids is accomplished within 10 min and the individual steroid peaks are quantitated by UV detection at 239 nm. This assay was examined for linearity, extraction efficiency, precision and potential interference by commonly used drugs. Plasma values of glucocorticoids are reported for samples obtained from human subjects as well as from rats. HPLC was also compared to RIA for the determination of plasma levels of corticosterone in the rat. Solid-phase extraction and assay by HPLC provides a rapid and specific method for the simultaneous determination of plasma glucocorticoids.

Cerebellar Hypoplasia in Neonatal Rats Caused by Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis virus, strain E-350, when inoculated intracerebrally in rats 1 to 7 days old, produces an acute destructive infection of the cerebellar cortex resulting in permanent cerebellar hypoplasia and ataxia. Several other arenoviruses may produce a similar lesion in neonatal rodents.

PATHOGENESIS OF CEREBELLAR HYPOPLASIA PRODUCED BY LYMPHOCYTIC CHORIOMENINGITIS VIRUS INFECTION OF NEONATAL RATS: 1. EVOLUTION OF DISEASE FOLLOWING INFECTION AT 4 DAYS OF AGE

Intracerebral inoculation of 4-day-old rats with LCM virus (Armstrong E-350 strain) resulted in a severe cerebellar necrosis and consequent ataxia. The pathogenesis of this disease was studied using virological, serological, and histological methods. Cerebellar necrosis occurred concomitantly with an acute and severe transitory choriomeningitis. While neural structures involved in postnatal replication were heavily infected, only the cerebellum consistently showed pathological changes. Necrosis was first evident in the granule cells, with secondary loss of Purkinje cells and white matter, but with sparing of the deep cerebellar nuclei. Brain virus reached high titers early after infection and gradually decreased finally to disappear 5 months after inoculation. Viremia was minimal and transitory, occurring within two weeks of infection. Although complement fixing antibodies developed, their relation to the cerebellar lesion is questionable since the pathology was well established prior to the occurrence of detectable levels of antibody. In addition, the acute phase of infection was associated with a retardation in growth rate, the consequence of which was a permanent impairment in development reflected by reduced brain and body weights.

Unequivocal demonstration of the hematogenous origin of brain macrophages in a stab wound by a double-label technique

Abstract We studied the origin of the phagocytic cells observed in the Neurol parenchyma in response to injury. Suckling albino rats were given repeated intraperitoneal injections of india ink to label circulating macrophages. Following this treatment, a discrete track lesion was produced in the cerebellar vermis by inserting into it a hypodermic needle which would deliver a minute amount of a second tracer, iron dextran. Electron-microscopic observation showed that 2 days later phagocytes within the lesion contained both iron dextran and carbon particles in their cytoplasm, indicating their hematogenous origin and the persistence of phagocytic activity.

Lack of b-cell participation in acute lymphocyte choriomeningitis disease of the central nervous system.

Abstract The pattern of acute lymphocytic choriomeningitis (LCM) virus-induced central nervous system (CNS) disease was studied in BALB/c mice rendered deficient in B cells and incapable of producing a humoral immune response. The CNS disease in mice treated from birth with goat anti-mouse μ-chain globulin (μ-suppressed mice) was clinically and histopathologically indistinguishable from that of controls. This result demonstrates that neither B cells nor antibody participate in the pathogenesis of acute LCM of the CNS. These observations support the view that the host cell-mediated response to viral antigens is solely responsible for acute CNS disease induced by the intracerebral inoculation of LCM virus.

VIRUS-INDUCED CELL-MEDIATED IMMUNOPATHOLOGICAL DISEASE

Publisher Summary This chapter explains virus-induced cell-mediated immunopathological disease. There are several important observations that provide a foundation for understanding virus-induced cell-mediated immunopathology. Several classes of viruses mature by budding through cellular membranes, thereby, acquiring their outer envelope. The principal antigens of such envelopes are virus-specified proteins and are therefore foreign to the host. Many enveloped RNA viruses are relatively or absolutely noncytocidal, so that infected cells may function and divide in an apparently normal manner while persistently carrying virus-coded neoantigens on their membranes. The immune response to enveloped viruses can result in the production of antibodies which, with complement, can lyse either intact virus or viable virus-infected cells. T cell-mediated cytolysis can be used as the basis for an in vitro assay of cell-mediated immunity. The occurrence of virus-induced immunopathological disease is rendered plausible by these considerations. However, the interaction of virus and immune response in vivo introduces a variety of additional complexities that are at present only partially understood.

Pathogenesis of LCM Disease in the Rat

Intracerebral inoculation of neonatal rats with LCM virus resulted in varying extents of central nervous system parenchymal pathology dependent upon host age at infection and upon virus strain. Lesions were confined to brain areas undergoing postnatal proliferation and migration, with the most striking involvement being a profound necrosis of most of the cerebellar cortex following infection of rats at 4 days of age. The immunopathological basis of this LCM virus-induced cerebellar pathology has been demonstrated both by preventing it with immunosuppression of normally susceptible animals and by eliciting it by adoptive immunization, with LCM virus-immune spleen cells, of infected animals not normally susceptible.

LYMPHOCYTIC CHORIOMENINGITIS VIRUS‐INDUCED CEREBELLAR HYPOPLASIA IN NEONATAL RATS *

Lymphocytic choriomeningitis (LCM) virus infections of mice following intracerebral inoculation have been extensively 9-11 The outcome of such infections has been shown to be age-de~endent.~ Neonatal mice have the ability to sustain an asymptomatic, life-long infection unaccompanied by detectable histologic changes in neuronal elements of the brain, but nevertheless carry high virus levels in this organ and other tissues. In contrast, older animals usually die following an acute convulsive diathesis that is thought to be mediated by a component of the host’s virus-specific immune response acting upon cells of the meninges, choroid plexus, and ependyma whose membranous surfaces have been altered by the infectious process.s Histologically there is a severe inflammatory reaction that is largely confined to these areas without morphological alteration of the cerebral parenchyma. Relatively few data exist on LCM virus infections of rats beyond what has already been reported,*. :’ which suggests that a detailed study of infection in this host might present an interesting comparison with previous studies employing mice. We now report the occurrence of an acute destructive process of the cerebellum following intracerebral inoculation of LCM virus into the neonatal rat. This striking pathological lesion, to our knowledge, has not been previously described.

Impairment in Auditory and Visual Function Follows Perinatal Viral Infection in the Rat

Acoustic startle reflexes are elicited by intense tone bursts but inhibited if weak bursts precede reflex elicitation. Rats were infected by intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) at birth. Compared to control animals, infected animals had higher elicitation and inhibition thresholds and showed recruitment at intense stimulus levels. Histopathology revealed both cochlear and retinal degeneration. Like some infectious agents in humans, perinatal exposure to LCMV in the rat yields a severe polysensory neuropathy.