Norman H. Bass

Postnatal development of bulk flow in the cerebrospinal fluid system of the albino rat: Clearance of carboxyl-[14C]inulin after intrathecal infusion

Car☐yl-[14C]inulin was infused into the spinal subarachnoid space of unanesthesized albino rats at 5, 10 and 30 postnatal days of age. Total cerebrospinal fluid volumes, estimated by infusions post mortem, were 122 ± 12 μl, 241 ± 24 μl and 250 ± 16 μl for 5-, 10- and 30-day-old rats, respectively. Measurements were made of steady-state rates at which inulin was removed from the cerebrospinal fluid system, constituting an estimate of the rate of cerebrospinal fluid formation. Values of 0.34 ± 0.03, 1.76 ± 0.18 and 1.88 ± 0.17 μl/min were found for 5-, 10- and 30-day-old rats, respectively. When inulin clearance was expressed as a percentage of the total fluid volume formed per minute, values of 0.28 ± 0.02, 0.73 ± 0.07,and0.75 ± 0.07 were found at 5, 10 and 30 postnatal days, respectively. Between 5 and 10 postnatal days of age, the total cerebrospinal fluid volume and rate of cerebrospinal fluid formation increased from 40 to 95% of 30-day-old values, correlating with morphological observations showing rapid maturation of the secretory epithelium of the choroid plexus. During early postnatal development, the formation of cerebrospinal fluid by the poorly differentiated columnar cells of the choroid plexus was greatly decreased and accompanied by sluggish bulk flow through the ventricles and subarachnoid spaces. It is hypothesized that, during this time, the inefficient ‘sink action’ of the cerebrospinal fluid system for extracellular space solutes in conjunction with an increased rate of entry of substances from the systemic circulation might increase the vulnerability of the developing brain to drugs and toxic agents.

Arachidonate‐induced cerebrovascular occlusion in the rat The role of platelets and aspirin in stroke

Unilateral cerebrovascular occlusion was produced in heparinized rats within seconds after injection of microgram quantities of sodium arachidonate into the internal carotid artery. Electroencephalographic activity over the affected cerebral hemisphere became attenuated, and cerebral blood flow decreased by half. A neurologic syndrome, including ipsilateral blindness and contralateral sensorimotor deficits, resulted from occlusion of the microvasculature by platelet thrombi. Although aspirin strongly inhibited arachidonate-induced platelet aggregation in vitro, the drug offered little protection against arachidonate-induced stroke.

Effects of hypothyroidism on the differentiation of neurons and glia in developing rat cerebrum.

Abstract Microchemical techniques and histologic examination were used to study the somatosensory area of developing cerebral cortex and subcortical white matter in rats made thyroid hormone deficient immediately after birth. The body weight of these animals failed to show the normal growth spurt, achieving levels which were only 40% of age-matched controls by 50 post-natal days. The endocrine imbalance initiated by a lack of thyroid hormone from the day of birth results in a metabolic derangement which disturbs the migration of glial cells from the subependymal zone into cortex between 10 and 50 days of post-natal life. During this same developmental period, the differentiation of neurons is impaired, resulting in decreased formation of axodendritic processes and synapses. Finally, in association with the pathologic development of axons and fewer numbers of well-differentiated interfascicular oligodendroglia, there is a failure of myelin formation. Hence, in rats made hypothyroid at birth, the cerebrum is subjected to drastic alterations in the normally continuous and interdependent process of neuron and glial cell differentiation.

Microchemical studies of postnatal development in rat cerebrum 2. Formation of myelin

AS A RESULT of electron microscopic studies, it is generally agreed that oligodendrocytes form myelin in the central nervous system.] Membranous connections between oligodendrocytes and external myelin lamellae and between axons and internal myelin lamellae have been found in the optic nerves,z cerebellum,3 and cerebrum4 of rat brain during postnatal development. Furthermore, glial cytoplasm is present between the myelin and axon and on the outer surface of the myelin spiraL6 These observations suggest that the oligodendrocytic membrane is not merely associated with the myelin sheath but that myelin is spirally wound, compact oligodendroglial cell membrane and cytoplasm. The myelin sheath has layers of radially arranged molecules of lipid alternating with circumferential molecules of protein.”,’ Electron micrographs show myelin as a repeating structure of three electron-dense lines: two “period lines” and one “interperiod line.”7ss The dark lines are regarded as protein and the two inteivening light areas as lipid. Cholesterol, cerebrosides, and proteolipid protein constitute a major part of the lipoprotein complex of myelin. High concentrations of these substances are found in areas of somatosensory cortex traversed by myelinated fibers in the adult rat, thus serving as quantitative biochemical indexes of cortical myeloarchitecture.9 Cholesterol constitutes about 30% of the total lipids in myelin fractions,l”.” and the sterolphospholipid molar ratio of myelin greatly exceeds that of brain mitochondria and of plasma membrane fractions from the muscle and liver.’ 2 Cerebrosides comprise nearly one-third of the total lipids of myelin and may be largely restricted to oligodendroglial and myelin membranes.“-” Proteolipid protein is uniquely concentrated in bovine myelin fractions,’OJ* and studies of the whole brain of the mouse and rat during development show a progressive rise in proteolipid concentration, paralleling the increasing mass of myelin.l:~-l” The present study of the somatosensory area of rat cerebrum during postnatal development shows changes in biochemical composition correlated with myelination. These data will serve as a base line for future investigations of experimental alterations of myelinogenesis.

Cortical alterations in zones of secondary epileptogenesis: A neurophysiologic, morphologic and microchemical correlation study in the albino rat

Abstract Microchemical techniques combined with histological and electroencephalographic observations were used to study a cortical zone of secondary epileptogenesis, produced in rats by the contralateral topical application of powdered cobalt. The results were compared with similar but non-epileptic cortical specimens from animals subjected to contralateral thermocoagulative lesions, and with normal controls. At 21 days, all animals showed a ‘mirror focus’ of persistent spike activity contralateral to the site of cobalt application. No epileptic activity was seen contralateral to the coagulative lesions. A suppression of cortical electrical activity was found over both cobalt and coagulative lesions, which showed, histologically, neuronal loss, glial proliferation, and neovascularization. Silver impregnation studies showed similar patterns of axonal degeneration resulting from both types of lesions. There was abundant degeneration surrounding the site of injury and degeneration of transcallosal projections to the contralateral cortical area. Contralateral and homotopic to both lesions, DNA, an index of cellularity, increased significantly only in layers V and VI, suggesting a possible glial proliferation in association with the axonal degeneration. Two biochemical abnormalities were found peculiar to the mirror focus, and possibly causally related to the epileptic process. RNA, an index of protein synthesizing ribosomal organelles, was reduced in all cortical layers. Ganglioside sialic acid, a neuronal membrane component, was strikingly increased in layers III through V, with a maximum elevation in layer IV. In non-epileptic cortex contralateral to coagulative lesions, the intralaminar profiles of these two biochemical components were similar to normal controls. These results suggest that a pathologic decrease of neuronal ribosomes, associated with either an elaboration of synaptic contacts or a functional disturbance of ganglioside-related ion transport may be causally related to the epileptic process.

Microchemical studies of postnatal development in rat cerebrum: I. Migration and differentiation of cells

THE BRAINS of most mammals at birth are functionally, structurally, and biochemically immature. After birth, the numerical density of neurons decreases, the number of glia increases, processes of neurons and glia proliferate, synapses form, and axons become myelinated. Postnatal maturation of the brain in various species may be completed in days, weeks, months, and even years. The albino rat is particularly suited for study of developing cerebral cortex because the brain is relatively undifferentiated at birth. The brain therefore can be studied at a time of development when in other species the organism is still in the uterus. The somatosensory isocortex of the albino rat was chosen for investigation partly because the fine structure of this area of cerebrum has been extensively determined by many workers.14 Furthermore, this region in the adult rat has been investigated biochemically in regard to the distribution of neurons, glia, and myelinated Neurochemists have assembled data on developing whole brain in many species, including man.7-11 Although of great value, these observations do not permit close morphological correlations because different regions of developing brain mature asynchronously. Microchemical techniques have made it practical to sample regions of brain in species as small as the rat.12J3 Quantitative biochemical indexes of histological structure have been found to reflect subtle tissue changes sometimes not seen with light microscopy.13-15 In the present study, DNA is used as an index of total num-

Neuronal compartmentation of ionic cobalt in rat cerebral cortex during initiation of epileptiform activity

Abstract An understanding of the mechanisms responsible for chronic epileptiform discharge induced by topical application of metallic cobalt to the cerebral cortex of the rat has been complicated by the progressive inflammation and necrosis of cortical tissue. In this study, ionic cobalt was transferred from a pipette on the pial surface into rat sensorimotor isocortex at a rate of 4 μg/min using an iontophoretic current of 3 μamp. Bilateral discharges of high voltage associated with brief bursts of ictal spike and polyspike activity were observed after transfer of 100 μg of cobalt. No histologic evidence of tissue damage was found by light microscopy. Quantitative and qualitative histochemical analysis revealed that cobalt was confined to the upper three cortical layers, occupying a circular area 1 mm in diameter on the pial surface. Although 75% of the heavy metal was found in layer I, neurons and their processes were intensely stained in layers II, and III. Hence, epileptiform discharge originated from neuronal aggregates which have a selective affinity for ionic cobalt and occupy a focal site in upper layers of sensorimotor cortex.

Mechanisms for the elimination of 5-hydroxyindoleacetic acid from brain and cerebrospinal fluid of the rat during postnatal development

Abstract Mechanisms employed for the elimination of 5-hydroxyindoleacetic acid (5-HIAA) from brain and cerebrospinal fluid (CSF) were studied in rats during early postnatal development. Probenecid was used to inhibit the removal of 5-HIAA by active transport mechanisms. In the 1- and 4-day-old animals, the major route for efflux of 5-HIAA from brain to blood was indirectly via CSF pathways, and was mediated, presumably at the choroid plexus, by an active transport mechanism possessing an efflux rate sufficient to remove all the 5-HIAA formed in the immature brain. The slow rate of bulk flow of CSF could account for the removal of only a small percentage of the 5-HIAA formed in brain, and active transport of the catabolite from brain capillaries could not be demonstrated. In contrast, the elimination of 5-HIAA via CSF pathways was of minor importance in the 30-day-old rat. More than 75% of the major metabolite of 5-hydroxytryptamine was removed directly from brain to blood by an active transport mechanism, presumably located at the glia-capillary interphase.

Postnatal development of mechanisms for the elimination of organic acids from the brain and cerebrospinal fluid system of the rat: Rapid efflux of [3H]para-aminohippuric acid following intrathecal infusion

Abstract Mechanisms for the elimination of organic acids from the brain and CSF system were studied during early postnatal development, by infusing tracer doses of [3H]para-aminohippuric acid into the spinal subarachnoid space of unanesthetized albino rats at 5 and 30 days of age. The rate of bulk flow in the CSF system of the infant rat, as measured by the elimination of [14C]inulin, was extremely slow, reflecting the presence of relatively undifferentiated cells composing the secretory epithelium of the choroid plexus. Nevertheless, [3H]PAH was found to be rapidly eliminated by a mechanism of active transport which was inhibited by high concentrations of 5-HIAA and systemically injected probenecid. In contrast, a carrier-mediated transport system was not found in the CSF system of the 30-day-old rat, and the efflux rate for the organic acid could be accounted for entirely by a markedly facilitated rate of bulk flow. In the brain of 30-day-old rats, another seemingly independent mechanism for the rapid elimination of organic acids to the peripheral circulation was found. This efflux, presumably localized at brain capillaries, was mediated exclusively by active transport which was inhibited by 5-HIAA and probenecid. A mechanism for carrier-mediated transport was not found in the brain of the 5-day-old rat, although an equally rapid efflux of PAH occured as a result of diffusion through relatively undifferentiated cerebral capillaries.

Microassay of diphenylhydantoin: Blood and regional brain concentrations in rats during acute intoxication

DIPHENYLHYDANTOIN (DPH) , first introduced by Merritt and Putnaml in 1938, has achieved widespread use for the treatment of convulsive disorders. Over the years, the drug has not only maintained its importance in the treatment of epilepsy but has been used to treat certain nonepileptic conditions such as cardiac arrhythmias2 and trigeminal neuralgia.3 The toxic manifestations of this drug on the nervous system are well known and include drowsiness, nystagmus, ataxia, and depression of deep tendon reflexes with alteration in nerve conduction.4-10 In the clinical management of patients on long-term treatment with DPH, frequent determination of blood levels is recommended for the following reasons: [ l ] patients are frequently unreliable in taking medication prescribed on a long-term basis,ll-ls [2] serum concentrations of DPH are variable in different patients on the same dosage,l*-l6 [3] clinical toxicity can be produced on low oral doses of the drug as a result of congenital catabolic enzyme deficiency, acquired hepatic dysfunction, or drugs interfering with DPH catabolism,ll [4] DPH can produce toxic clinical manifestations which may be confused with other diseases of the central nervous system,l7 and [5] chronically sustained toxic levels of the drug may possibly result in irreversible damage to the central nervous system with degeneration of Purkinje cells.lOJS-zO Although various spectrophotometric methods of DPH assay have been rep0rted,l~*~l-~4 they have either been too time consuming for use in most clinical laboratories or lacked the specificity necessary to prevent chemical interference with many commonly used drugs. In 1966, Wallace and co-workers25 reported an ultraviolet spectrophotometric assay for the determination of serum levels of DPH that did not require preliminary separation from other drugs. The assay was based on the chemical conversion of diphenylhydantoin to benzophenone, resulting in a product with a welldefined ultraviolet absorption spectrum and high molar absorptivity. However, the assay procedure, as originally devised, required 10 ml. of whole blood or serum for a single determination, thus limiting its clinical application. The present study describes a micromodification of the DPH assay reported by Wallace26 that is both rapid and reproducible, employing only 1 to 2 ml. of whole blood or 500 mg. wet weight of tissue. This new method has been used to study the relationship between concentrations of DPH in blood and various brain regions of albino rats during acute intoxication.