Heterogeneity affecting outcome from acute stroke therapy: making reperfusion worse.

BACKGROUND: Stroke patients are heterogeneous not only with respect to etiology but also in terms of preexisting clinical conditions. Approximately one fifth of patients with acute stroke are hyperglycemic and/or have had a recent infectious or inflammatory condition. Summary of Review-- Experimental research indicates that these factors can alter and accelerate the evolution of stroke and reperfusion injury, although these effects are complex and some may have a favorable impact. Both conditions involve activation of inflammatory and reactive oxygen mechanisms. In addition, hyperglycemia has concomitant deleterious vascular and metabolic effects that worsen infarct size and encourage hemorrhagic transformation in reperfusion models. Clinical data are less extensive but in general support an adverse impact on outcome. CONCLUSIONS: After examining these data in detail, we concluded that the presence of these clinical conditions could assist in identification of those at increased risk for complications of reperfusion therapy. Furthermore, consideration of these factors may provide a rational basis for combination therapy and improve the clinical relevance of experimental stroke models.

Electrochemical monitoring of superoxide anion production and cerebral blood flow: effect of interleukin-1 beta pretreatment in a model of focal ischemia and reperfusion.

Conditions associated with systemic infection, such as endotoxinemia, are known to increase the levels of pro-inflammatory cytokines such as interleukin (IL)-1 in the central nervous system. Systemic infection has been shown to be a common preexisting condition in patients with stroke. To examine a possible consequence of systemic infection, we used a novel electrochemical technique, which combines measurement of cerebral blood flow with measurement of superoxide anion concentrations, to examine the effect of pretreatment of pial vasculature with a proinflammatory cytokine, IL-1 beta, on cerebral blood flow and superoxide anion concentration in a rat model of middle cerebral artery occlusion and reperfusion. In addition, neutrophil recruitment was measured using an immunohistochemical technique. Our results indicate that exposure of pial and cerebral vasculature to IL-1 beta significantly accelerates recruitment of neutrophils, reduces cerebral blood flow, and increases superoxide anion concentration at the pial surface during reperfusion. These results support the idea that prior exposure of brain vasculature to IL-1 beta results in acceleration of cerebrovascular injury by accelerating recruitment of neutrophils, which secrete superoxide anion, during reperfusion. This finding has possible implications for the treatment of stroke with reperfusion agents in patients with preexisting infections.

Punctate midline myelotomy for the relief of visceral cancer pain.

OBJECT: This study offers clinical support for the concept that neurosurgical interruption of a midline posterior column pathway by performing a punctate midline myelotomy (PMM) provides significant pain relief without causing adverse neurological sequelae in cancer patients with visceral pain refractory to other therapies. METHODS: A PMM of the posterior columns was performed in six cancer patients in whom visceral pain had been refractory to other therapies. The cause of the visceral pain was related to residual, progressive, or recurrent local cancer or postirradiation effects. Clinical efficacy of the procedure was examined by comparing patient pain ratings and narcotic usage pre- and post-PMM. Follow-up periods ranged from 3 to 31 months. Examination of the results indicates a significant reduction in pain ratings as well as a significant reduction in daily narcotic use. No adverse neurological effects were observed. One spinal cord has been recovered for postmortem examination. CONCLUSIONS: These findings provide corroborating clinical evidence for the existence of a newly recognized midline posterior column pathway that mediates the perception of visceral pelvic and abdominal pain. Preliminary data indicate that significant pain relief can be obtained following PMM with minimal neurological morbidity and suggest that the procedure may provide an alternative treatment modality for cancer-related pain in patients in whom adequate pain control with narcotics cannot be achieved or narcotic side effects cannot be tolerated.

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.

The 21-aminosteroid U-74389G reduces cerebral superoxide anion concentration following fluid percussion injury of the brain.

We examined the effects of the 21-aminosteroid antioxidant U-74389G (16-desmethyl-tirilazad) on the concentration of extracellular superoxide anion following fluid percussion traumatic brain injury (TBI) measured by a cytochrome c-coated electrode and on local cerebral perfusion (CBFld) measured by laser Doppler flowmetry (LDF). U-74389G in a dose of 3 mg/kg reduced superoxide anion concentrations 60 min after TBI significantly but had no significant effect on CBFld. These results indicate that reduction of CBF after TBI can be dissociated from superoxide anion production. Persistent ischemia may limit neuroprotection efficacy and may contribute to divergent outcome results in clinical and animal trials using agents to modify reactive oxygen species.

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.

Plasma nerve growth factor access to the postnatal central nervous system.

The accessibility of macromolecules to the postnatal mammalian central nervous system is a subject of controversy and importance. One of the biologically important macromolecules in neural development, nerve growth factor (NGF), is thought to be unable to cross the blood-brain barrier in postnatal mammals. In this study, I125 labeled 7S NGF was systemically administered to postnatal rats and was found to accumulate in the brain in significant levels. In addition, brains from saline perfused rats contained intact 7S NGF as determined by gel autoradiography. Radiolabeled NGF was found to be localized to specific areas corresponding to p75 NGF receptor distribution in the hippocampus and cortex by autoradiography. Circulating plasma NGF has access to the developing postnatal neuraxis where it may exert a humoral trophic function.

Neuronal toxicity by macrophages in mixed brain cell culture is augmented by antineuronal IgG and dependent upon nitric oxide synthesis.

We used mixed brain cell cultures derived from dissociated neonatal rat cerebella to study interactions between mononuclear phagocytes and brain cells under various conditions. We found that activated macrophages were capable of selectively killing neurons, leaving other cells undisturbed. Moreover, this activity was dependent upon nitric oxide production and, to a weaker extent, upon the NMDA receptor but not upon tumor necrosis factor. Macrophage-mediated neuronolysis was augmented by one of two anti-neuronal antibodies studied.

Poor reliability of immunocytochemical localization of IgG in immersion-fixed tissue from the central nervous system.

The effect of fixation technique and post mortem-to-fixation interval in immersion-fixed tissue from the central nervous system on immunocytochemical staining for the presence of an immunoglobulin was determined in mice. Immersion-fixed tissue was found to be inferior to perfusion-fixed tissue for immunocytochemical staining of this serum protein. Unlike what has been observed for other antigens, the quality of staining for IgG in immersion-fixed tissue decreased to unacceptable levels if the post mortem-to-fixation interval was increased to more than a few hours. This effect may be secondary to the rapid post-mortem disintegration of the blood-brain barrier and a resulting diffusion of serum proteins into surrounding tissue from the vasculature.

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.

Time course of penetration of xenogeneic IgG into the central nervous system of the neonatal rat: an immunohistochemical and radionuclide tracer study.

We investigated the penetration of rabbit IgG into the brain and spinal cord of newborn rat pups and compared them to mature rats using immunohistochemical and radionuclide techniques. Following intraperitoneal injection with rabbit IgG and perfusion fixation at various intervals, rabbit IgG was found to penetrate into the parenchyma of the neuraxis of neonatal rats and to establish much higher concentrations than was found in mature rats. Immunohistochemical studies revealed no evidence of significant extravasation of serum proteins from the vasculature. For any given survival interval studied, there was no significant difference in appearance and intensity of immunohistochemical staining or the amount of radiolabeled IgG measured between different areas of the brain and spinal cord. Electrophoresis of radioactive proteins extracted from the brains of neonatal rats injected with radiolabeled IgG confirmed that IgG penetrates into the brain parenchyma as the whole molecule. These results validate the use of systemically administered rabbit IgG for studies of brain development in neonatal rats. They also have implications for the pathogenesis of congenital central nervous system anomalies in which maternal autoimmune IgG may affect brain development after being transferred to the fetus or neonate.

Penetration of IgGs into the neuraxis of the neonatal rat.

The permeability of the mammalian blood-brain barrier to macromolecules during prenatal and postnatal development is a controversial issue. We tested the possible access of xenogeneic antibodies to the neuraxis by examining neural tissue of neonatal rats 48 h after intraperitoneal injection of rabbit IgGs, using a modification of the peroxidase-antiperoxidase (PAP) technique. The results of the present study indicate diffuse immunoreactivity for rabbit IgGs in the parenchyma in all regions of the neuraxis in neonatal rats. This work suggests that immunoglobulins, both maternal and isogeneic, may affect the nervous system during prenatal and postnatal development.

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: 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.

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.

Axonal transport of monoclonal antibodies.

Three monoclonal antibodies against rat brain synaptosomes, produced by conventional hybridoma techniques, were screened for their ability to undergo uptake and axonal transport in vivo. Injections of ascitic fluid or of purified immunoglobulin G (IgG) were made into the vitreal chamber of the eye in anesthetized rats to test for anterograde transport in retinal afferents to the contralateral superior colliculus. Retrograde transport by facial nucleus motoneurons was evaluated after injections of antibody into the mystatial vibrissal skin and musculature. Transported immunoglobulins were localized in tissue sections using a modification of the peroxidase-antiperoxidase technique. One monoclonal antibody, S-2C10, was found to undergo anterograde transport in retinal ganglion cells and retrograde axonal transport in facial motoneurons. Transported immunoglobulins were detectable even after injections of dilute antibody solution (0.01-0.05% IgG), and the uptake-transport process for this antibody appeared saturable. Two other antibodies tested, S-4E9 and S-1G10, exhibited the ability to undergo retrograde transport, but only after injections at relatively high antibody concentrations (greater than or equal to 1.0% IgG). Neither of these antibodies was shown to undergo anterograde transport. Following retrograde transport in motoneurons, the S-2C10 antibody was localized in neuronal perikarya, proximal dendrites, and the adjacent neuropil of the facial motor nucleus. In contrast, the S-4E9 and S-1G10 antibodies were localized in punctate granules within neuronal cell somata following transport. The findings suggest that the uptake-transport process for the S-2C10 antibody is mediated by adsorptive endocytosis following binding of the antibody to a plasma membrane component (or components) present in somadendritic and nerve terminal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

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)