Roderic H. Fabian

In vivo detection of superoxide anion production by the brain using a cytochrome c electrode.

A cytochrome c-coated platinized carbon electrode was utilized to detect superoxide generated by the brain during hypoxia/hypercarbia, focal ischemia, and reperfusion and following fluid percussion brain injury with and without hemorrhagic hypotension and reperfusion in the rat. All three of these forms of brain injury were associated with an increase in the superoxide signal. The cytochrome c electrode proved to be sensitive and responsive enough for minute-by-minute measurement of superoxide generation by brain tissue.

Transneuronal transport of lectins

Axonal and transneuronal transport of the plant lectins wheat germ agglutinin (WGA), Pisum sativum agglutinin (PSA), Lens culinaris agglutinin (LCA), soybean agglutinin (SBA), peanut agglutinin (PNA), Concanavalin A agglutinin (Con A), and Ulex europeus agglutinin (UEA) were examined and compared using an immunocytochemical staining method. WGA, which binds to N-acetylglucosamine and sialic acid carbohydrate residues, and the 3 mannose binding lectins (Con A, PSA and LCA) were found to undergo retrograde transport to the facial nucleus after injection into the facial muscles, and anterograde transport to the optic tectum after injection in the vitreous, and to the spinal trigeminal nucleus caudalis after injection into the mystatial vibrissae. SBA showed a slight tendency to be transported retrogradely, but not in the anterograde direction, whereas UEA and PNA were not axonally transported in any of these systems. All lectins which were transported in the anterograde direction labeled neuronal somata in their respective terminal fields indicating that transneuronal transport had taken place. Axonal and transneuronal transport of the lectins appears to be dependent upon their respective carbohydrate affinities. Transneuronal transport which can be demonstrated for certain lectins indicates that mechanisms exist whereby neurons exchange large molecules which could be involved in mediating trophic and other influences on target cells.

Intraneuronal IgG in the central nervous system Uptake by retrograde axonal transport

The uptake of immunoglobins by CNS neurons was studied in rats. Rats were injected IP with solutions containing large amounts of rabbit IgG. Immunocytochemical staining of sections of the neuraxis revealed uptake of rabbit IgG by motor neurons of the CNS with axons projecting outside of the blood-brain barrier, including ventral horn motor neurons and cranial nerve motor nuclei neurons as well as in neurons projecting to the hypothalamus and area postrema. Staining was also noted in certain large neurons of the reticular formation and in Purkinje cells, as well as diffusely in the hypothalamus, area postrema, the pia mater, and associated vasculature and larger penetrating vessels. Uptake of rabbit IgG by lumbar spinal cord motor neurons projecting to the sciatic nerve was prevented by ligation of the sciatic nerve. These experiments support the hypothesis that certain central neurons take up immunoglobins from the periphery by retrograde axonal transport. The function of this process is not known, but it may have significance for the pathogenesis of motor and autonomic neuropathies and neuronopathies.

Perivascular nitric oxide and superoxide in neonatal cerebral hypoxia-ischemia

Decreased cerebral blood flow (CBF) has been observed following the resuscitation from neonatal hypoxic-ischemic injury, but its mechanism is not known. We address the hypothesis that reduced CBF is due to a change in nitric oxide (NO) and superoxide anion O(2)(-) balance secondary to endothelial NO synthase (eNOS) uncoupling with vascular injury. Wistar rats (7 day old) were subjected to cerebral hypoxia-ischemia by unilateral carotid occlusion under isoflurane anesthesia followed by hypoxia with hyperoxic or normoxic resuscitation. Expired CO(2) was determined during the period of hyperoxic or normoxic resuscitation. Laser-Doppler flowmetry was used with isoflurane anesthesia to monitor CBF, and cerebral perivascular NO and O(2)(-) were determined using fluorescent dyes with fluorescence microscopy. The effect of tetrahydrobiopterin supplementation on each of these measurements and the effect of apocynin and N(omega)-nitro-L-arginine methyl ester (L-NAME) administration on NO and O(2)(-) were determined. As a result, CBF in the ischemic cortex declined following the onset of resuscitation with 100% O(2) (hyperoxic resuscitation) but not room air (normoxic resuscitation). Expired CO(2) was decreased at the onset of resuscitation, but recovery was the same in normoxic and hyperoxic resuscitated groups. Perivascular NO-induced fluorescence intensity declined, and O(2)(-)-induced fluorescence increased in the ischemic cortex after hyperoxic resuscitation up to 24 h postischemia. L-NAME treatment reduced O(2)(-) relative to the nonischemic cortex. Apocynin treatment increased NO and reduced O(2)(-) relative to the nonischemic cortex. The administration of tetrahydrobiopterin following the injury increased perivascular NO, reduced perivascular O(2)(-), and increased CBF during hyperoxic resuscitation. These results demonstrate that reduced CBF follows hyperoxic resuscitation but not normoxic resuscitation after neonatal hypoxic-ischemic injury, accompanied by a reduction in perivascular production of NO and an increase in O(2)(-). The finding that tetrahydrobiopterin, apocynin, and L-NAME normalized radical production suggests that the uncoupling of perivascular NOS, probably eNOS, due to acquired relative tetrahydrobiopterin deficiency occurs after neonatal hypoxic-ischemic brain injury. It appears that both NOS uncoupling and the activation of NADPH oxidase participate in the changes of reactive oxygen concentrations seen in cerebral hypoxic-ischemic injury.

Superoxide anion production during reperfusion is reduced by an antineutrophil antibody after prolonged cerebral ischemia

Neutrophils may be involved in the pathophysiology of reperfusion injury following cerebral ischemia. One potential mechanism of reperfusion injury by neutrophils is through production of the superoxide anion. We hypothesized that, due to progressive endothelial damage during ischemia, neutrophil activation would be more prominent after longer periods of ischemia prior to reperfusion. Thus, neutrophils would contribute more to pathological processes such as superoxide anion formation after longer than after shorter periods of ischemia. A reversible middle cerebral artery occlusion model in rats was employed and superoxide anion concentration was measured with a cytochrome c coated electrode placed on the cortical penumbral region. Occlusion times were varied from 60 min to 2 h, and neutrophils were inhibited with an antiCD18 antibody administered prior to occlusion. Neutrophil accumulation and reduction with antibody treatment was confirmed immunohistochemically. Superoxide anion (O2*-) concentration was detected during the hours following 60 min of occlusion, and increased further with 2 h of occlusion. Treatment with the antiCD18 antibody had no effect on O2*- concentration during reperfusion in the 60-90 min occlusion groups, but O2*- concentration was significantly lower in the antiCD18 antibody treated group than in the control group during reperfusion after 120 min of ischemia. The antibody also reduced cortical neutrophil accumulation in the 120 min ischemia group. These results indicate for the first time that superoxide production by neutrophils becomes more important with longer periods of ischemia, and other quantitatively less important sources of superoxide predominate with shorter periods of ischemia. This phenomenon may explain some of the variation seen between different models of ischemia with different durations of ischemia when targeting reactive oxygen species, and supports an approach to combination therapy to extend the therapeutic window and reduce the deleterious effects of reperfusion.

Uptake of plasma IgG by CNS motoneurons Comparison of antineuronal and normal IgG

We studied the uptake of antisynaptosomal and nonspecific IgG by ventral horn motoneurons in rats, using immunohistochemical and radionuclide techniques. Plasma antisynaptosomal IgG is taken up to a much greater extent than nonspecific IgG by motoneurons that project outside the blood-brain barrier, as is radiolabeled antisynaptosomal IgG injected intramuscularly. Competition with unlabeled antisynaptosomal IgG inhibits the uptake of radiolabeled antisynaptosomal IgG. By contrast, competition with unlabeled nonspecific IgG does not inhibit the uptake of radiolabeled nonspecific IgG. These results support the hypothesis that certain neurons in the CNS take up IgG from the systemic circulation, and that IgG which binds to elements of the synaptic plasma membrane is taken up in greater amounts than nonspecific IgG through a process of adsorptive endocytosis at the nerve terminus. An increase in intraneuronal IgG may serve as an index of the action of antineuronal IgG at the presynaptic membrane.

Intraneuronal IgG in the central nervous system

The rat central nervous system was examined immunocytochemically for the presence of endogenous IgG. Examination of representative sections of the neuraxis revealed specific staining for IgG in the pia mater and pial vasculature, the ependyma, and diffusely in the hypothalamus and area postrema where the blood-brain barrier is permeable to large molecules. In addition, intraneuronal staining for IgG was noted in specific nuclei including the ventral horn nuclei and intermediolateral nuclei of the spinal cord, the dorsal motor nucleus of the vagus, the nucleus ambiguous, the motor nucleus of the trigeminal, the hypoglossal, facial, and oculomotor nuclei, nuclei projecting to the pituitary and area postrema, and Purkinje cells. The uptake of immunoglobins by these cell groups may have important implications for the pathogenesis of motor and autonomic neuropathies and neuropathies.

Uptake of antineuronal IgM by CNS neurons Comparison with antineuronal IgG

We examined the axonal transport of monoclonal IgM that binds to Thy 1.1, a glycoprotein component of the neuronal and synaptic plasma membrane, in the rat, using immunohistochemical techniques. IgM immunoreactivity appeared in neurons 1 to 2 days following injection in their terminal fields. Several neural pathways supported retrograde axonal transport of IgM, including facial nucleus, hypoglossal nucleus, and thalamic projections. Although the IgM studied had a greater affinity for Thy 1.1 than the IgG, IgM axonal transport was more difficult to detect than axonal transport of IgG, suggesting that IgM undergoes retrograde axonal transport less readily than IgG. An increase in intraneuronal IgM may serve as an index of the action of antineuronal IgM at the presynaptic membrane, and intraneuronal IgM may be directly involved in the pathogenesis of some types of motor neuron disease and neuropathy.

Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters

Significance Mechanistic studies of nontoxic hydrophilic carbon cluster nanoparticles show that they are able to accomplish the direct conversion of superoxide to dioxygen and hydrogen peroxide. This is accomplished faster than in most single-active-site enzymes, and it is precisely what dioxygen-deficient tissue needs in the face of injury where reactive oxygen species, particularly superoxide, overwhelm the natural enzymes required to remove superoxide. We confirm here that the hydrophilic carbon clusters are unreactive toward nitric oxide radical, which is a potent vasodilator that also has an important role in neurotransmission and cytoprotection. The mechanistic results help to explain the preclinical efficacy of these carbon nanoparticles in mitigating the deleterious effects of superoxide on traumatized tissue. Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O2•−) dismutase-like properties yet were inert to nitric oxide (NO•) as well as peroxynitrite (ONOO−). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO2 and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O2•− to O2 by PEG-HCCs at >20,000 s−1. The major products of this catalytic turnover are O2 and H2O2, making the PEG-HCCs a biomimetic superoxide dismutase.

Axonal transport of antibodies to subcellular and protein fractions of rat brain

Experiments examined the feasibility of using the axonal transport of antibodies as a possible means to characterize nerve membrane composition and the fate of internalized macromolecules. Polyspecific antibodies were generated in rabbits against rat brain synaptosomal and microsomal subcellular fractions and against wheat germ agglutinin-binding proteins isolated by lectin affinity chromatography. Antisera were injected into the vitreal chamber of the eye and into the facial musculature of anesthetized rats to test, respectively, for anterograde transport in retinotectal neurons and for retrograde transport in facial motoneurons. Control injections of preimmune serum were made into the opposite side. After survival for 4-168 h, animals were perfused and the axonally transported rabbit immunoglobulins detected in frozen sections of the brainstem using a modified peroxidase-antiperoxidase immunocytochemical procedure. Antisera against all 3 classes of neuronal antigens contained antibodies that underwent retrograde axonal transport. No evidence of anterograde transport was seen. Neurons containing retrogradely transported immunoglobulins exhibited punctate as well as diffuse staining of the cytoplasm and proximal dendrites, exclusive of the nucleus. Following retrograde transport of antibodies to the synaptosomal fraction, staining of the neuropil around motoneurons was also observed, suggesting transcellular transport of these antibodies. Concentrations of injected antibodies as low as 1% of whole antiserum led to detectable retrograde transport. Increasing concentrations of antibodies above the amount in whole antiserum did not increase the intensity of staining in retrogradely labeled neurons, suggesting saturation. The findings support the view that antibodies to neural membranes are taken up and transported by binding to specific sites on nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)