Kenneth Maiese

Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global ischemia.

We recently demonstrated that reactive astrocytes express NADPH diaphorase activity, a marker for Nitric Oxide Synthase, following transient global ischemia (Neuroscience Letters 154: 125-128). There has been little evidence that astrocytes express Nitric Oxide Synthase or produce NO (nitric oxide) in vivo; although in vitro experiments have shown that cultured astrocytes can produce NO. To determine whether reactive astrocytes express inducible form of NOS (iNOS) in vivo, we studied the pathological changes of rat hippocampus by immunohistochemistry after 10 minutes of transient global ischemia, which results in the selective delayed death of CA1 pyramidal cells and marked gliosis in the CA1 subfield. In the normal hippocampus, astrocytes express neither NADPH diaphorase activity nor iNOS. After ischemia, the temporal and spatial pattern of iNOS, NADPH diaphorase, and GFAP are very similar, indicating that reactive astrocytes express iNOS. Double staining for NADPH diaphorase and GFAP, or iNOS and GFAP confirmed that reactive astrocytes express both NADPH diaphorase activity and iNOS immunoreactivity. These changes were observed three day after ischemia and increased in prominence from one week to one month. The staining pattern of OX42, an antibody that recognizes both microglia and macrophages, is spatially and temporally distinct from the pattern of NADPH diaphorase and iNOS staining. Thus, we conclude that transient global ischemia induces iNOS primarily in reactive astrocytes. This increase in NOS expression and, presumably, NO production by reactive astrocytes may play a role in the process of delayed neuronal death or in the remodeling responses that occur after ischemic damage.

Expression of the neural form of nitric oxide synthase by CA1 hippocampal neurons and other central nervous system neurons.

Nitric oxide can act as a neurotransmitter and a retrograde modulator of synaptic transmission, but uncontrolled nitric oxide synthase activity has been associated with neural degeneration. Although earlier studies using immunohistochemistry, in situ hybridization, and NADPH-diaphorase staining had suggested that nitric oxide synthase is not expressed in the CA1 neurons of the hippocampus, we have recently demonstrated that NADPH-diaphorase activity can be detected in CA1 neurons of the hippocampus. To confirm that this diaphorase activity reflects nitric oxide synthase, we have developed a more sensitive in situ hybridization procedure, and an RNase protection assay to detect message for constitutive nitric oxide synthase, the form constitutively expressed in many neurons. Message for constitutive nitric oxide synthase is expressed in the hippocampus, and it is localized to neural cell layers CA1, CA3, the dentate gyrus and some displaced neurons, but not to CA2. Expression of constitutive nitric oxide synthase message in the CA1 region was lost when pyramidal neurons died due to transient forebrain ischemia, supporting the conclusion that CA1 pyramidal cells express constitutive nitric oxide synthase. Although constitutive nitric oxide synthase message is strongly expressed in CA3 and the dentate gyrus, there is little diaphorase activity in these cells, suggesting that there may be post-transcriptional controls that limit constitutive nitric oxide synthase expression in some cells. Message for constitutive nitric oxide synthase is also present in a number of other regions, including the amygdala, several hypothalamic nuclei, the cerebellum, the olfactory bulb, two distinct regions of the perirhinal cortex, the subthalamic nuclei, a neuronal layer in the retrosplenial granular cortex, the lateral geniculate nucleus, the presubiculum, the inferior colliculus, the superior colliculus, the pedunculopontine tegmental nucleus, and scattered individual neurons in the cortex, hippocampus and brainstem. These studies support a role for nitric oxide in multiple regions of the central nervous system. In particular, nitric oxide synthase, the enzyme responsible for the synthesis of nitric oxide, is expressed in the CA1 region of the hippocampus, where there is evidence that nitric oxide may play a major role in long-term potentiation. CA1 hippocampal neurons are an example of a population of neurons that express constitutive nitric oxide synthase but are very sensitive to excitotoxicity and ischemic insults.

Reduction in Focal Cerebral Ischemia by Agents Acting at Imidazole Receptors

Treatment with the α2-adrenergic antagonist idazoxan (IDA) can provide protection from global cerebral ischemia. However, IDA also recognizes another class of receptors, termed imidazole (IM) receptors, which differ from α2-adrenergic receptors and are responsible for the hypotensive actions of some centrally acting agents such as the oxazole rilmenidine (RIL). We therefore sought to determine whether RIL, an agent highly selective for IM receptors, offered protection from focal cerebral ischemia elicited in rat by ligation of the middle cerebral artery (MCA). We compared the effects of RIL with the effects of IDA and the selective non-IM α2-antagonist SKF 86466 (SKF). In addition, we examined whether the neuroprotective effects of RIL and IDA could be attributed to changes in local CBF (LCBF). The MCA was occluded and animals either received immediate administration of drug while arterial pressure was maintained for 1 h or had local CBF increased to 200% of control for 1 h by hypercapnia or hypertension. RIL elicited a significant dose-dependent preservation of tissue to 33% of control at optimal dose (0.75 mg/kg). IDA (3 mg/kg) significantly reduced the size of ischemic infarction by 22%. In contrast, SKF (15 mg/kg) as well as doubling of LCBF did not preserve ischemic tissue. We conclude that both RIL and IDA can reduce focal ischemic infarction but that the mechanism does not appear secondary to antagonism of α2-adrenergic receptors or elevation of LCBF. Occupation of IM receptors, either in the ischemic zone or at remote brain sites, may be responsible for neuroprotection of RIL and IDA.

Reactive astrocytes express NADPH diaphorase in vivo after transient ischemia.

In the hippocampus, ten minutes of transient global ischemia results in the death of CA1 pyramidal cells after a period of one to three days. The neurons in the CA1 region constitutively express NADPH-D (NADPH diaphorase activity). In contrast, astrocytes in the hippocampus do not normally express NADPH-D; but a population of reactive astrocytes (GFAP+ cells) begin to express of NADPH-D one day after transient global ischemia. NADPH-D is thought to be a histological marker for Nitric Oxide Synthase (NOS), the enzyme that is responsible for the synthesis of NO, a potent neurotoxin. We suggest that this increase in NADPH-D/NOS expression is an important element in the sequence of changes that occurs after ischemia, and that NO derived from reactive astrocytes or from neurons may play a causal role in neural cell death after ischemia in the hippocampus.

T‐cell lymphoma in the CNS Clinical and pathologic features

T-cell lymphoma may involve the CNS as either a primary or secondary neoplasm. This report describes 8 patients with either primary or secondary T-cell malignancies in the CNS. Five patients presented with symptoms and signs of CNS disease that included seizures, visual impairment, cranial nerve palsies, sensory and motor deficits, gait ataxia, and paraparesis. Three of them had primary parenchymal CNS lymphoma, and 2 had epidural lymphoma that originated in adjacent bone marrow. Three patients were neurologically asymptomatic, but had leptomeningeal tumor and focal parenchymal infiltration at postmortem examination. Histologically, 4 lymphomas were large cell, 3 were mixed large and small cell, and 1 could not be classified by the working formulation for non-Hodgkins lymphomas. The clinical and pathologic manifestations of T-cell lymphoma in the CNS may be diverse. This report demonstrates that neurologic abnormalities may be the presenting signs of either primary CNS or systemic T-cell lymphoma.

Nitric oxide : a downstream mediator of calcium toxicity in the ischemic cascade

Loss of cellular calcium homeostasis or the production of nitric oxide (NO) have been cited as possible mechanisms that may contribute to neuronal degeneration during ischemia. We therefore examined whether cellular calcium blockade, using the agent HA1077, was protective during anoxia in hippocampal neuronal cell cultures, and whether the in vitro effects of this drug were linked to the NO pathway. Administration of the agent during anoxia was neuroprotective in neuronal cell culture. In contrast, HA1077 did not protect hippocampal neurons during NO exposure. In addition, inhibition of NO synthesis in conjunction with HA1077 application during anoxia did not significantly increase survival beyond the maximum protection afforded by HA1077 alone. These results suggest that calcium may be an initial messenger in the ischemic cascade, but that subsequent neuronal degeneration is dependent upon the NO pathway.

Effect of acute and chronic arecoline treatment on cerebral metabolism and blood flow in the conscious rat

Treatment with the muscarinic agonist arecoline improves memory retention in patients with Alzheimers disease (AD). In animal models, arecoline selectively increases local cerebral glucose utilization (LCGU). We examined (1) whether these focal increases in metabolism were coupled to local cerebral blood flow (LCBF) and (2) whether the effect of arecoline on LCGU and LCBF was dependent upon duration of drug administration. In groups of young Fischer-344 rats, LCGU and LCBF were determined in 59 brain regions by the [14C]2-deoxyglucose and the [14C]iodoantipyrine autoradiographic methods following either the acute administration of arecoline (2 mg/kg and 15 mg/kg) or the chronic three week administration of arecoline (50 mg/kg/day). In general, LCBF correlated closely with LCGU following arecoline 2 mg/kg administration, but heterogeneous regions were present. Following treatment with arecoline 15 mg/kg, the two parameters became uncoupled with LCBF increasing disproportionately in relation to LCGU. Coupling between LCBF and LCGU was preserved during chronic arecoline treatment (50 mg/kg/day) but some regions, such as the hippocampus, were uncoupled with LCGU increasing to a greater extent than LCBF. Thus, we demonstrate that acute and chronic administration of arecoline can differentially modulate LCBF and LCGU. Since clinical administration of arecoline can improve cognitive function in patients with AD, understanding the ability of arecoline to selectively alter LCBF and LCGU in regions such as the hippocampus may offer insight into the pathophysiology of AD and provide direction for the development of definitive therapy for neurodegenerative disorders.

Protein kinase C modulates the protective ability of peptide growth factors during anoxia

Neuronal degeneration following exposure to anoxia and nitric oxide (NO) may be modulated by peptide growth factors and the activity of signal transduction systems. Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) are neuroprotective during anoxia and NO toxicity. Signal transduction systems that activate protein kinase C (PKC) can be detrimental to neurons and mediate the toxic effects of anoxia and NO. We therefore examined whether PKC was involved in the protective effects of bFGF and EGF during anoxia. After exposure to anoxia, approximately 20-30% of hippocampal neurons survive. In contrast, chronic down-regulation of PKC activity prior to anoxia increases hippocampal neuronal cell survival to approximately 75%. Yet, this protective effect of inhibition of PKC activity was not present with the application of peptide growth factors during anoxia. Combined inhibition of PKC activity and application of the peptide growth factors bFGF or EGF was detrimental to the hippocampal neurons during anoxia. Neuronal survival during anoxia was 68 +/- 2% with bFGF and 79 +/- 3% with EGF but decreased to 49 +/- 7% (bFGF) and 44 +/- 2% (EGF) with PKC down-regulation. Addition of the growth factors with the agent H-7, an inhibitor of PKC activity, also decreased neuronal survival during anoxia. In addition, the protective effects of the growth factors during anoxia were lessened to a greater degree with the activation of PKC, decreasing hippocampal neuronal survival for bFGF to 23 +/- 2% and for EGF to 31 +/- 3%.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Neuronal Vulnerability to Ischemia by Peptide Growth Factors and Intracellular Second Messenger Systems: The Role of Protein Kinase C and the cAMP Dependent Protein Kinase

Abstract The vulnerability of neurons to the damaging events that occur during a period of ischemia can be modulated by a wide range of peptide growth factors and by agents that modify intracellular signal transduction systems both in vivo and in vitro. Analysis of the molecular mechanism of these protective effects can help to establish how signaling cascades influence the sensitivity of neurons to ischemia. We have established two in vitro models of ischemia and used these to explore the ability of peptide growth factors to confer protection on neural cells. Using the rat PC12 cell line, we have established a model of ischemia that includes a combination of hypoglycemia and anoxia. We also established an in vitro model of ischemia using primary cultured hippocampal neurons that has been used to study both anoxia and nitric oxide toxicity. In both models, peptide growth factors are neuroprotective. Although each of the growth factors we have studied is known to activate PKC, its activation does not mimic the effects of the peptide growth factors in either model. Indeed, down-regulation of PKC or pharmacologic inhibition of PKC made PC12 cells resistant to ischemic damage. Likewise, down-regulation of PKC or inhibition of PKC activity made hippocampal neurons more resistant both to ischemia and nitric oxide toxicity. In contrast, activation of the cyclic adenosine monophosphate (cAMP) dependent protein kinases (PKA) confers protection in both model systems. The effects of PKA activation were not additive with those of the growth factors. The effects of the growth factors were equivalent in PC12 lines that contained normal levels of the PKA, or were deficient in this enzyme. Thus, there appears to be two signal transduction pathways that contribute to neural protection in both PC12 cells and hippocampal neurons. One pathway is under the control of peptide growth factors, and a second can be initiated by activation of PKA. In PC12 cells, the protective effects of nerve growth factor (NGF) are mediated through the trk receptor and are dependent on the activation of the N-kinase, a member of the MAP kinase family and a key regulator of NGF-dependent differentiation. Thus, in two quite distinct model systems, PKC appears to have a deleterious effect on neuronal survival, whereas activation of the cAMP-dependent protein kinase appears to be neuroprotective. A significant fraction of the neuronal death that occurs in hippocampal neurons during anoxia is caused by the generation of nitric oxide. Inhibition of nitric oxide synthase (NOS) reduced the extent of neuronal death, and additions of agents that generate nitric oxide is neurotoxic. Growth factors can protect against nitric oxide toxicity, and one of the major mechanisms used by peptide growth factors to protect these neurons against ischemia is to make them resistant to nitric oxide that is generated during periods of ischemia. If nitric oxide is an important mediator of ischemic damage in vivo, the source of nitric oxide must be identified. We therefore examined the expression of both the constitutive form of nitric oxide synthase (cNOS) and the inducible form of nitric oxide synthase (iNOS) after ischemia. Using in situ hybridization and the histochemical nicotinamide-adenine-dinucleotide phosphate-reduced form (NADPH) diaphorase reaction, we were able to demonstrate that pyramidal neurons in CA1, CA3, CA4, and granule cells in the dentate gyrus express cNOS. In contrast to a number of instances where neurons that express NOS are resistant to neurodegeneration, neurons in the CA1 region of the hippocampus express cNOS, but are very vulnerable to ischemia. Although NOS is not normally expressed in astrocytes, transient ischemia induces the expression of iNOS in astrocytes, as indicated by both immunohistochemistry and NADPH diaphorase histochemistry. Thus, the synthesis of nitric oxide by either neurons or astrocytes might contribute to cell death during ischemia.