Stress induced morphological microglial activation in the rodent brain: involvement of interleukin-18.

The present study investigated the possibility that acute stress might activate microglial cells. Wistar rats were exposed to 2 h period of restraint combined with water immersion stress prior to brain analysis by immunohistochemistry with OX-42, a marker of complement receptor CR3. A single session of stress provoked robust morphological microglial activation in the thalamus, hypothalamus, hippocampus, substantia nigra and central gray. These effects appeared as early as at 1 h of exposure and were further intensified at 2 h. Morphological activation was not accompanied with changes in markers of functional activation or of inflammation including interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS). Similar results were obtained with mice where the effects of stress were compared in animals null for interleukin-18 (IL-18 KO), a cytokine previously demonstrated to be modulated by stress and to contribute to microglia activation. The results demonstrated significant reduction of stress-induced microglial activation in IL-18 KO mice. The present study reports evidence that physical/emotional stress may induce morphological microglial activation in the brain and this activation is in part mediated by interleukin-18.

Interleukin-18 mRNA expression in the rat pituitary gland.

The present study investigated the expression of IL-18 mRNA under several stimuli, and molecular structures of IL-18 mRNA of the rat pituitary. Real-time PCR demonstrated that IL-18 mRNA, highly expressed in anterior pituitary, significantly increased following stress and adrenalectomy. In situ hybridization combined with immunohistochemistry demonstrated that corticotrope cells expressed IL-18 mRNA. RACE and sequence analysis demonstrated that pituitary IL-18 mRNA possesses five new exons at the upstream of exon 1 and between exon 1 and exon 2, indicating the preferential usage of promoter 1. The present study suggests that IL-18 in the corticotrope cells may play some roles in stress responses.

Extracellular H+ stimulates the expression of c-fos/c-jun mRNA through Ca2+/calmodulin in PC12 cells.

Evidence has accumulated that an increase in extracellular protons stimulates the transmembrane mechanism to induce various intracellular responses, such as the expression of c-fos and c-jun. In the present study, we aimed to obtain evidence that an increase in extracellular protons induces expression of c-fos/c-jun mRNA in PC12 pheochromocytoma cells of rats. We found that the c-fos/c-jun mRNA expression increased when extracellular pH was decreased gradually from 7.40 to 7.20 and that there was a significant correlation between extracellular pH values and the expression of c-fos/c-jun mRNA. To determine whether the Ca2+/calmodulin system subserves the H+-induced expression of c-fos/c-jun, Ca2+/calmodulin inhibitor trifluoperazine was added to PC12 cells. We found that trifluoperazine inhibited the expression of the H+-induced c-fos/c-jun mRNA by 30-35%. In contrast, trifluoperazine did not inhibit the expression of phorbol-induced c-fos/c-jun mRNA. These results indicate that an increase in extracellular protons induces the expression of c-fos/c-jun mRNA, and this expression is mediated partly by the Ca2+/calmodulin system.

Cell-type specific signal transduction and gene regulation via mitogen-activated protein kinase pathway in catecholaminergic neurons by restraint stress.

It has been demonstrated that tyrosine hydroxylase (TH) gene is easily regulated in the CNS as well as peripheral nervous systems by stressful conditions. The stimuli, such as stress or reserpine administration, significantly increased the TH gene in noradrenergic neurons in the locus ceruleus (LC), but not in dopaminergic neurons in the substantia nigra (SN). To explore the molecular mechanisms governing differential TH gene regulation in catecholaminergic cells, the present study investigated the regulation of immediate early gene (c-Fos), transcription factors (pCREB, CREB binding protein [CBP]), mitogen-activated protein (MAP) kinases (phospho-extra-cellular regulated kinase [pERK]1/2, phospho-p38 MAP kinase [p-p38 MAPK], phospho-c-Jun N-terminal kinase [pJNK]) in the LC and SN in control conditions and in response to 2 h restraint stress (RS). Significant induction of c-Fos expression was observed in the LC, but not in the SN. In addition, pERK1/2 significantly increased following 2 h RS specifically in the LC, but not in the SN. No significant change was observed in p-p38 MAPK and pJNK. The expression of c-Fos and pERK1/2 preceded the upregulation of TH in the LC. Furthermore, pCREB and CBP also increased in the LC in response to 2 h RS. The induction of c-Fos prior to TH, in conjunction with the upregulation of pCREB and CBP in the LC, suggests that activator protein 1 and CRE transcription sites in the TH gene may be involved in the cell-type specific activation in the stress response, at least, by pERK1/2.

Interleukin-18 null mice show diminished microglial activation and reduced dopaminergic neuron loss following acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment.

Recent reports have revealed an involvement of microglial cells in dopaminergic neurodegeneration. In the present study, we tested the hypothesis that interleukin-18 (IL-18) plays a role in the microglial activation. The present study investigated microglial activation and dopaminergic neurodegeneration in substantia nigra pars compacta (SNpc) following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in wild type (WT) and IL-18 knockout (KO) mice. The number of dopaminergic neuron loss in WT mice was significantly decreased 7 days after MPTP treatment compared with IL-18 KO mice. In WT mice microglial activation occurred in the SN at 1 day after MPTP treatment, progressively increased within the SNpc until 7 days post MPTP, and subsided by 14 days. In contrast, in IL-18 KO mice microglial activation occurred in the SN at 1 day post-MPTP, and decreased by 7 days, earlier than in WT mice. The lesser microglial activation and dopaminergic neurodegeneration in the SNpc following MPTP treatment in WT indicates the possibility that IL-18 may participate in microglial activation and dopaminergic neurodegeneration.

Temporal and sequential analysis of microglia in the substantia nigra following medial forebrain bundle axotomy in rat.

Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis after transection of the medial forebrain bundle. We have assessed the temporal and sequential activities of microglia in these events by examining the complement-3 (OX-42), major histocompatibility complex class II antigen presentation (OX-6) and phagocytic activity (ED1), and correlating these indicators with dopaminergic neuronal loss. Microglia in the ipsilateral substantia nigra pars reticulata evinced activation morphology at 12 h postaxotomy. Phagocytic microglia apposed dying dopaminergic neurons in the pars compacta starting at 3 days postlesion; their number increased through 14 days and slowly decreased. Nuclear chromatin condensation and significant loss of tyrosine hydroxylase-positive dopaminergic neurons occurred around 7 days postlesion. In contrast to microglial expression of interleukin-1beta and inducible nitric oxide synthase at the axotomy site, nigral microglia were interleukin-1beta and inducible nitric oxide synthase-negative. Consistently, RNase protection assays showed that interleukin-1beta and inducible nitric oxide synthase transcripts in nigra were equivocal. The present data support the idea that phagocytosis of axotomized neurons by activated microglia is not limited to dead neurons but includes dying neurons probably without cytotoxic effects of inflammatory substances, such as interleukin-1beta or nitric oxide.

In vitro study of H+-sensitive neurons in the ventral medullary surface of neonate rats.

We hypothesized that the direct stimulus of the central chemoreceptor neurons is the CO2/H+-induced change in intracellular pH (pHi). If it is true, pHi responses during hypercapnic stimulation should be exhibited in the central chemoreceptor neurons in the ventral medullary surface (VMS) and some neurons in the CO2/H+ sensitive regions such as the nucleus tractus solitarii of the medial dorsal medulla (MDM). To test this hypothesis, the cultured VMS and MDM neurons (control) derived from one day-old neonate rats were labeled with H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and were exposed to perfusate of various pHs. The H+-sensitive neurons were determined by a rapid decrease in the intracellular BCECF fluorescence intensity. In almost all the MDM neurons (99.6%) and 94% of the VMS neurons, the intracellular BCECF fluorescence intensity remained unchanged when the extracellular pH (pHo) was decreased. In contrast, in 0.4% of the MDM neurons (8/1800) and in 6% of the VMS neurons (111/1800), the intracellular BCECF fluorescence intensity decreased when the pHo was decreased from 7.4 to 7.2. This subpopulation of MDM and VMS neurons were considered to be H+-sensitive neurons. The H+-sensitive neurons in the VMS showed positive immunoreactivity to glutamate (57%, 17/30) and glutamic acid decarboxylase (23%, 7/30), but no immunoreactivity to choline acetyltransferase, tyrosine hydroxylase, phenylethanolamine N-methyltransferase, somatostatin, serotonin and substance P. These results indicate that the H+-sensitive neurons are present specifically in the VMS, and are mainly glutamatergic and GABAergic.

H+-sensitivity of cultured neurons from the dorsomedial and ventrolateral medulla of neonate rats.

The H+-sensitivity of neonate rat cultured neurons derived from the dorsomedial medulla (DMM) containing the nucleus tractus solitarii and the ventrolateral medulla (VLM) was determined by H+-sensitive fluorescent probe BCECF-AM and immunohistochemical methods. Against an extracellular pH as low as 7.2-7.3, H+-sensitivity was verified in 2.6% of the DMM neurons (46/ 1800) and 2.1% of the VLM neurons (38/1800). This H+-sensitive neurons of the DMM were immunoreactive to glutamate (52.4%) and glutamic acid decarboxylase (GAD) (28.6%), while those of the VLM were immunoreactive to glutamate (66.7%) and GAD (33.3%). There was no immunoreactivity to tyrosine hydroxylase, phenylethanolamine-N-methyltransferase or choline acetyltransferase in the H+-sensitive neurons are present in the DMM and VLM besides the ventral medullary surface, the site of the central chemoreceptors.

A case of dyskinetic cerebral palsy resembling post-anoxic action myoclonus.

Myoclonus after brain anoxia is more commonly observed in adults. Perinatal anoxia, however, on rare occasions causes myoclonus as the main neurologic abnormality. In this paper we described a case of cerebral palsy complicated by chorea and tremulous movements of the foot in which there were no risk factors except perinatal asphyxia. The patient had unique symptoms, action myoclonus associated with the movements of hyperkinésie volitionnelle. This case illustrated two points. One is that abnormal involuntary movements attributed to perinatal brain insults had changed as the brain matured. The other is that some movements were very similar to those of the post-anoxic action myoclonus, commonly seen in patients who sustain brain damage in adulthood.

A sporadic case of very slow progressive leukodystrophy involving the cerebellar peduncles.

We describe a patient with infantile onset leukodystrophy involving the cerebellar peduncles. She had mild mental retardation, spastic diplegia and mild cerebellar ataxia. The peripheral nerves seemed to be normal. The characteristic MRI findings in this case were extensive lesions of the white matter involving the cerebellar peduncles. In addition there was ventricular enlargement with a markedly decreased volume of the white matter and a hypoplastic corpus callosum. The clinical and laboratory findings imply that the white matter lesions in this patient were the result of delayed myelination rather than demyelination. The patient was evaluated for known metabolic and degenerative diseases, but no abnormalities were observed. Her symptoms and neuroimaging findings did not fit the criteria for any defined leukodystrophy.

Psychosine cytotoxicity toward rat C6 glioma cells and the protective effects of phorbol ester and dimethylsulfoxide: implications for therapy in Krabbe disease.

Psychosine cytotoxicity was tested as to its effects on rat C6 glioma cells. At a low concentration--below 40 microM--psychosine appeared to stimulate cell proliferation. Above the concentration range of 40 microM-60 microM, however, it showed a cytotoxic effect. When phorbol ester (PDB) or dimethylsulfoxide (DMSO) was supplemented to cultures being exposed to psychosine, the total number of live cells, protein content and CNPase activity dramatically increased as compared with the levels in cultures treated with psychosine alone. The results of these basic studies suggest another approach as to therapy for globoid cell leukodystrophy.

Transient, unilateral, isolated hypoglossal nerve palsy.

We report a boy with isolated hypoglossal nerve palsy that recovered within twelve weeks. Investigations of the cervix, base of the skull, medulla and hypoglossal nerve by CT-scan and MRI did not show any abnormal findings. There are a few reports concerning isolated unilateral hypoglossal nerve palsy with benign course. Although we could not establish the cause of our patient's disease, an awareness of this condition should save such patients from unnecessary invasive studies.

Monozygous twin with polymicrogyria and normal co-twin.

Monozygous twinning has been associated with brain lesions such as holoprosencephaly, hydranencephaly, multicystic encephalomalacia, and porencephaly. Polymicrogyria is rare but has been reported in autopsied cases. We present a monozygotic twin with complicating twin-twin transfusion syndrome, the donor exhibiting normal psychomotor development and the recipient psychomotor delay associated with the brain anomaly, polymicrogyria. The brain anomaly might be due to blood pressure instability or a circulatory disturbance resulting from increased coagulability due to polycythemia.

Magnetic resonance imaging in three children with kernicterus.

The incidence of kernicterus has been greatly reduced by effective monitoring and treatment for hyperbilirubinemia. Findings on magnetic resonance imaging (MRI) in patients with kernicterus are characteristic. This study presents three cases of possible kernicterus without typical symptoms but with MRI features consistent with kernicterus. These cases suggest that kernicterus can develop, especially in preterm infants, in the presence of relatively low levels of bilirubin and the absence of obvious acute symptoms. Therefore assessing the risk of kernicterus may be difficult in the neonatal period. In addition, MRI findings at the posteromedial border of the globus pallidus in patients with athetotic cerebral palsy are strong evidence of brain damage caused by kernicterus.

Enhanced magnetic resonance imaging of leptomeningeal angiomatosis.

We present two patients with unilateral occipital gyriform calcification and seizures. Gyriform or serpentine calcification as revealed by computed tomography (CT) scan is rare and is a characteristic finding of Sturge-Weber syndrome (SWS) and celiac disease (CD). These patients had neither the facial nevus flammeus or neurological deficits characteristic of SWS, nor the gastrointestinal symptoms characteristic of CD. CD is often accompanied by cerebral occipital calcification indistinguishable from that of SWS. We demonstrate the presence of cerebral leptomeningeal angiomatosis (LA) by Gadolinium-DTPA-enhanced magnetic resonance imaging (MRI) but could not detect LA by either CT scanning or angiography. It has been reported that contrast-enhanced MRI is useful to detect LA in SWS. However, we found no reports of enhanced MRI in patients with SWS without facial angioma. If future studies can demonstrate the absence of cortical enhancement by contrast-enhanced MRI in CD with cerebral calcifications, enhanced MRI would become an important tool for differentiating CD from SWS.

[A case of spastic paraparesis, mental retardation and thinning of corpus callosum].

There are several reports of the patients who have peculiar clinical presentation, that is, spastic paraparesis, mental disturbance and thinning of corpus callosum. We presented a sporadic case with similar symptoms. He was born by normal vaginal delivery without problems from consanguineous parents. His development had delayed since his infantile period. He started to have spasticity of the legs at the age 13 years and it worsened with advancing age. On examination at age 19, he was mentally slow, his full-scale IQ being 35. The tone in the arms was slightly increased and marked in the legs. The finger to nose test caused mild dysmetria. Rest of the neurological examinations were normal. MRI demonstrated characteristic thinning of corpus callosum, dominant in anterior portion. In addition, it showed wide cavum vergae and cavum septi pellucidi, and abnormal high intensity in periventricular white matter. The findings of MRI were possibly suggestive of the static developmental disturbance of brain.

[Central neurogenic hyperventilation in an awake patient with a primary cerebral lymphoma].

We described clinical and neuropathological findings of a case of primary cerebral lymphoma with central neurogenic hyperventilation (CNH). A 54-year-old awake woman with a primary cerebral lymphoma presented hyperventilation for two weeks. Arterial blood gas showed severe respiratory alkalosis; PH 7.603, PaCO2, 10.5 mmHg, PaO2 129.8 mmHg, HCO2 10.4 mmol/L, BE -8.0, O2SAT 98.9%. Rebreathing from a paper bag, and intravenous administration of diazepam and sodium bicarbonate failed to alter the respiratory pattern. Consecutive CAT scans indicated that CNH didn't occur when the tumor extensively invaded the cerebral cortex, cerebellum, thalamus and basal ganglia but was initiated when the lymphoma invaded the brain stem. Pathological study showed lymphoma cells invaded the cerebral cortex, cerebellum, thalamus and basal ganglia severely, and the brain stem moderately and recently. Contrary to the cases reported by Plum, Lange and Bateman, the lower medulla was also involved. Possible mechanisms for CNH are discussed in relation to the pathological findings and consecutive CAT scan findings.

[The neuroendocrinological examinations in mentally retarded boy with self-injurious behavior (SIB) and the effect of naloxone, clomipramine and sulpiride on SIB].

The neuroendocrinological examinations were performed in mentally retarded 7-year-old boy with severe self-injurious behavior (SIB). The thyroid-stimulating hormone (TSH) and prolactine responses to TRH showed lower in the basal and peak levels compared with those in the age matched control group. It is suggested that there may be an enhanced dopaminergic activity. The effects of naloxone, clomipramine and sulpiride on the frequency of SIB were investigated in this case. The frequency of SIB was reduced by sulpiride; a dopamine antagonist. These data suggest dopaminergic supersensitivity and hypothalamic dysregulation as a mechanism underlying SIB.

[A patient with epilepsy presenting developmental regression after seizures--consideration of the mechanism from the findings of CT-scan and SPECT].

We report a 6-month-old female infant with frequent brief seizures who subsequently showed developmental regression. CT-scans right after her seizures revealed enlargement of bilateral subdural space more on the right side with an evidence to suggest destruction of the blood brain barrier in enhanced CT-scan, CT-scan, one month after the onset, showed diffuse brain atrophy. 123I-SPECT, which was performed 11 days after cessation of her seizures, still showed diffuse hypoperfusion predominantly in the right temporal area, and it returned to normal 3 months after the onset. We discussed the mechanisms of developmental regression in this case from the neuroradiological findings.

[SPECT (single photon emission computed tomography) findings in 4 cases of neuronal migration disorders].

We report SPECT findings in four children with various types of neuronal migration disorders. SPECT of a patient with heterotopia showed a protrudent abnormal shadow into the centrum semiovale, the density of which was same as that of normal gray matter. The function and the metabolism of heterotopia appear same as those of the normal gray matter. SPECT of a patient with pachygyria showed decreased blood flow in the areas of thickened cortex. This finding seems to indicate hypofunction of those lesions. SPECT of a patient with hemimegalencephaly showed an increased blood flow in the ipsilateral hemisphere and decreased blood flow in the contra-lateral hemisphere. In the literature, increased contents of neuronal elements in the affected site and arterio-venous shunt with cardiac failure have been reported in hemimegalencephaly. These pathologies may be related to our SPECT findings. SPECT of a patient with cortical dysplasia showed extensive lesions of decreased blood flow which was more wide spread than abnormal lesions detected by MRI. SPECT may be more sensitive than MRI to detect cortical dysplasias.