Almost all degenerative diseases of the CNS are associated with chronic inflammation. A central step in this process is the activation of brain mononuclear phagocyte cells, called microglia. While it is recognized that healthy neurons and astrocytes regulate the magnitude of microglia-mediated innate immune responses and limit excessive CNS inflammation, the endogenous signals governing this process are not fully understood. In the peripheral nervous system, recent studies suggest that an endogenous 'cholinergic anti-inflammatory pathway' regulates systemic inflammatory responses via a7 nicotinic acetylcholinergic receptors (nAChR) found on blood-borne macrophages. These data led us to investigate whether a similar cholinergic pathway exists in the brain that could regulate microglial activation. Here we report for the first time that cultured microglial cells express a7 nAChR subunit as determined by RT-PCR, western blot, immunofluorescent, and immunochistochemistry analyses. Acetylcholine and nicotine pre-treatment inhibit lipopolysaccharide (LPS)-induced TNF-a release in murine-derived microglial cells, an effect attenuated by a7 selective nicotinic antagonist, a-bungarotoxin. Furthermore, this inhibition appears to be mediated by a reduction in phosphorylation of p44/42 and p38 mitogen-activated protein kinase (MAPK). Though preliminary, our findings suggest the existence of a brain cholinergic pathway that regulates microglial activation through a7 nicotinic receptors. Negative regulation of microglia activation may also represent additional mechanism underlying nicotine's reported neuroprotective properties.

Background and Purpose-Intravenously delivered human umbilical cord blood cells (HUCBC) have been previouslyshown to improve functional recovery of stroked rats. To extend these findings, we examined the behavioral recovery and stroke infarct volume in the presence of increasing doses of HUCBC after permanent middle cerebral artery occlusion (MCAO). Methods-Rats were subjected to MCAO and allowed to recover for 24 hours before intravenous infusion of 10 4 up to 3 to 5ϫ10 7 HUCBC. Behavioral tests (spontaneous activity, step test, elevated body swing test) were performed 1 week before MCAO and at 2 and 4 weeks after HUCBC infusion. On completion of behavioral testing, animals were euthanized and brain infarct volumes quantified. HUCBC were identified by immunofluorescence for human nuclei and by polymerase chain reaction (PCR) using primers specific for human glycerol 3-phosphate dehydrogenase. Results-At 4 weeks after infusion, there was a significant recovery in behavioral performance when 10 6 or more HUCBC were delivered (pϭ0.001 to pϭ0.05). Infarct volume measurements revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (10 7 cells, pϽ0.01; 3 to 5ϫ10 7 cells, pϽ0.05). Moreover, HUCBC were localized by immunohistochemistry and PCR analysis only in the injured brain hemisphere and spleen. Conclusions-These results extend previous observations of HUCBC infusion in the MCAO rat stroke model by demonstrating a dose relationship between HUCBC, behavioral improvement, and neuronal sparing.

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When human umbilical cord blood cells (HUCBCs) are administered intravenously after a middle cerebral artery occlusion, they reliably produce behavioral and anatomical recovery, and protect neural tissue from progressive change. However, our results indicate that the cells do not exert their effects by engraftment in the peri-infarct region, even though they migrate to the site of injury. The objective of the present study was to determine if the cells induce recovery by decreasing inflammation. We used a combination of in vivo and in vitro studies to show that HUCBCs decrease inflammation in the brain after stroke and thereby enhance neuroprotection. After stroke and transplantation, there was a decrease in CD45/CD11b- and CD45/B220-positive (+) cells. This decrease was accompanied by a decrease in mRNA and protein expression of pro-inflammatory cytokines and a decrease in nuclear factor kappaB (NF-kappaB) DNA binding activity in the brain of stroke animals treated with HUCBCs. In addition to modulating the inflammatory response, we demonstrate that the cord blood cells increase neuronal survival through non-immune mechanisms. Once thought of as "cell replacement therapy," we now propose that cord blood treatment in stroke reduces inflammation and provides neuroprotection. Both of these components are necessary for effective therapy.

Sleep and wakefulness, as well as time of day and night, are important considerations in proper characterization of seizure types and epilepsy localization. These findings may contribute to a better understanding of the mechanisms of nonrandom distribution of seizures, and may provide information for individualized treatment options.

Although considered an immunologically privileged site, the central nervous system (CNS) can display significant inflammatory responses, which may play a pathogenic role in a number of neurological diseases. Microglia appear to be particularly important for initiating and sustaining CNS inflammation. These cells exist in a quiescent form in the normal CNS, but acquire macrophage-like properties (including active phagocytosis, upregulation of proteins necessary for antigen presentation, and production of proinflammatory cytokines) after stimulation with inflammatory substances such as lipopolysaccharide (LPS). Recent studies have focused on elucidating the role of neurons in the regulation of microglial inflammatory responses. In the present study, we demonstrate, using neuron-microglial cocultures, that neurons are capable of inhibiting LPS-induced tumor necrosis factor-alpha (TNF-alpha) production by microglia. This inhibition appears to be dependent on secretion of substances at axon terminals, as treatment with the presynaptic calcium channel blocker omega-conotoxin abolishes this inhibitory effect. Moreover, we show that conditioned medium from neuronal cultures similarly inhibits microglial TNF-alpha production, which provides additional evidence that neurons secrete inhibitory substances. We previously demonstrated that the transmembrane protein-tyrosine phosphatase CD45 plays an important role in negatively regulating microglial activation. The recent characterization of CD22 as an endogenous ligand of this receptor led us to investigate whether neurons express this protein. Indeed, we were able to demonstrate CD22 mRNA and protein expression in cultured neurons and mouse brain, using reverse transcriptase-polymerase chain reaction and antibody-based techniques. Furthermore, we show that neurons secrete CD22, which functions as an inhibitor of microglial proinflammatory cytokine production.

SUMMARYPurpose: To evaluate the relationship of sleep/awake and circadian patterns to generalized seizures. Methods: Charts of 1,044 consecutive pediatric epilepsy patients undergoing video-electroencephalography (EEG) monitoring (vEEG) over 5 years were reviewed: 962 patients were excluded due to focal epilepsy (556), nonepileptic recorded events (217), missing data (125), age over 21 years (59), and no recorded events or seizures (10). Seizure semiology of recorded seizures with generalized onset on EEG was classified according to the International League Against Epilepsy (ILAE) seizure semiology terminology, and analyzed based on occurrence during day (6 a.m. to 6 p.m.) or night and on their relationship to wakefulness and sleep, with calculated occurrence in 3-h time blocks throughout 24 h. Statistical analysis was performed with binomial testing. Key Findings: Three-hundred sixteen generalized seizures were analyzed in 77 children. Mean age was 6.4 years ± 5.4 (range 0.33-20 years), including 50.6% girls. Tonic and tonic-clonic seizures were more frequently seen in sleep, whereas all other generalized semiologic seizure types occurred more frequently out of wakefulness. Clonic seizures had two peaks: (6-9 a.m.) and (noon to 3 p.m.) in wakefulness. Absence seizures occurred predominantly in wakefulness, (9 a.m. to noon and 6 p.m. to midnight). Atonic seizures occurred predominantly in wakefulness (noon to 6 p.m.). Myoclonic seizures occurred in wakefulness (6 a.m. to noon). Epileptic spasms had two peaks: (6-9 a.m. and 3-6 p.m.) in wakefulness. Significance: Circadian pattern and sleep-wake patterns are important considerations in characterization of generalized seizure types. Recognition and characterization of individual diurnal seizure patterns offer new diagnostic and therapeutic options, including EEG or long-term video EEG monitoring scheduling, differential (day/night) medication dosing, and a better understanding of pathophysiologic mechanisms underlying circadian patterns of epilepsy. KEY WORDS: Epilepsy monitoring, Circadian patterns, Generalized seizures, Semiology, Epilepsy.The daily periodicity of seizures in patients with epilepsy was reported in the early neurology literature (LangdonDown & Brain, 1929). Although seizures may be perceived by patients as unpredictable events, the occurrence of seizures is not entirely random (Quigg, 2000). Gowers observed that ''diurnal'' seizures cluster at certain times of the day, namely upon awakening and in the late afternoon, and that ''nocturnal'' seizures tend to occur mainly at bedtime and in the early morning hours before awakening (Langdon-Down & Brain, 1929;Griffiths & Fox, 1938). Janz (1962) noted that up to 45% of patients with primarily generalized tonic-clonic seizures had nocturnal seizures. Recent findings both in animal models and in human partial epilepsy suggest a direct circadian influence on seizures of limbic origin (Quigg et al., 1998. Patients with frontal lobe epilepsy typically have partial seizures arising from sleep (Bazil & Walcz...

Filgratism (granulocyte colony stimulating factor, G-CSF)-mobilized peripheral blood progenitor cells (PBPCs) have replaced bone marrow (BM) as a preferred source of autologous stem cells, in light of the faster hematologic recovery and lesser supportive care requirement exhibited by PBPC transplants. Other hematopoietic stem cells, like the human umbilical cord blood-derived stem cells (hUCBs), and nonhematopoietic stem cells have been shown to improve motor function in rodent models of injury and degenerative disease. In the present study we transplanted either G-CSF-mobilized PBPCs or hUCBs in rats 24 h after permanent middle cerebral artery occlusion (MCAO), and assessed their behavioral abnormalities in spontaneous activity and spontaneous motor asymmetry. In both transplanted groups of rats we observed a significant reduction of the stroke-induced hyperactivity compared with nontransplanted, stroked animals. In addition, transplantation of G-CSF PBPC and hUCB cells prevented the development of extensive motor asymmetry. Our findings raise the possibility that PBPCs could provide a novel transplantation therapy to treat stroke.