Corticosteroids limit microglial activation occurring during acute stress.

Our previous studies demonstrated that exposure of animals to acute stress immediately induced morphological microglial activation in the brain. Here we investigated the effects of adrenal corticoids on microglial activation following acute stress. We compared microglial activation in vivo in adrenalectomized (ADX), Sham-operated (SHM), and adrenalectomy plus corticosterone (CORT) administered rats exposed to a 2-h period of acute water restraint stress. We found that: (1) acute stress induced microglial activation in SHM rats; (2) acute stress robustly enhanced microglial activation in ADX rats; (3) CORT treatment significantly reduced the effects of adrenalectomy. Thus, while acute stress has the ability to activate microglia, the magnitude of activation is negatively regulated by CORT. Glucocorticoids may serve as an important endogenous suppressive signal limiting neuroinflammation that might otherwise occur during stress.

Immunological responses of astroglia in the rat brain under acute stress: interleukin 1 beta co-localized in astroglia.

In previous studies, we demonstrated that acute stress induces microglial activation, without inducing any inflammatory responses; however, the effect of acute stress on astroglia, another glial cell subtype in the brain, remains to be elucidated. We determined the effect of acute stress on astroglia, particularly in terms of morphological changes and inflammatory properties. In contrast to microglia, the morphology of astroglia was not altered following a 2-h period of acute stress. Interestingly, the number of astroglia immunoreactive to interleukin 1 beta (IL-1ß) significantly increased in several brain regions including the hippocampus, hypothalamus, amygdala, and periaqueductal gray following the acute stress. Confocal microscopy revealed that IL-1ß is exclusively co-localized in astroglia, and not in neurons or microglia. The present study demonstrates that exposing rats to acute stress increases IL-1ß immunoreactivity in astroglia in specific regions of the brain, and the mechanism of astroglial response to acute stress clearly differs from that of microglial response. Thus, astroglia may play important roles in neuroimmunomodulation through IL-1ß during times of acute stress.

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.

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.

[Cranioplasty for isolated trigonocephaly with developmental disorder].

We reported 50 cases of mild to moderate trigonocephaly (most isolated type) treated by cranioplasty. All of them had clinical symptoms such as severe hyperactivity, speech delay, inability to communicate with others, self-mutilation (head banging), irritability, temper tantrum and mental retardation. Pre-operative CT scan and MRI showed no abnormal findings in the brain except for constricted frontal lobes. The 3 D-CT scan showed the most important diagnostic findings: a ridge of the metopic suture and narrow anterior fossa. TcECD SPECT was performed on 43 patients, and demonstrated in 31 cases some degree of decreased cerebral blood flow (CBF), mainly in the bilateral frontal lobes. Post-operatively, most patients improved to some degrees. The results were compared to those of trigonocephaly patients without cranioplasty. The operated group showed better improvement in the above clinical symptoms, especially, hyperactivity, indifference to others, understanding of verbal communication, self-mutilation, irritability and temper tantrum. The post-operative SPECT represented the increased CBF in 30 out of the 31 cases. MRI and CT scan revealed expanded frontal lobes. Thus, cranioplasty may alleviate the symptoms of patients with mild to moderate trigonocephaly and developmental disorders.

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.

Frontal lobe dementia with abnormal cholesterol metabolism and heterozygous mutation in sterol 27-hydroxylase gene (CYP27).

Of the primary dementing disorders that cause frontotemporal dementia, the best-known is Pick disease. We report on a 44-year-old woman with progressive frontal lobe dementia and spastic paraplegia. Examination revealed increased serum levels of cholestanol with abnormal cholesterol metabolism and a heterozygous mutation of the sterol 27-hydroxylase gene (CYP27). Biochemical findings were compatible with cerebrotendinous xanthomatosis (CTX); however, the clinical manifestations were very dissimilar. To our knowledge, a symptomatic carrier of this mutation among CTX patients has not been reported. We speculate that the present patient has a previously undescribed neurodegenerative disease related to abnormal cholesterol metabolism with this heterozygous mutation.

Tissue-specific expression of rat IL-18 gene and response to adrenocorticotropic hormone treatment.

IL-18 is a pleiotropic cytokine also proposed to have a role in modulating immune function during stress. Initially found in immune cells, IL-18 mRNA is detectable in several tissues including the cells of the zona reticularis and the zona fasciculata of the adrenal cortex, where its levels are elevated by acute stress or adrenocorticotropic hormone treatment. In the present study, we compared the expression of IL-18 in the adrenal cortex with that of spleen and duodenum, two other IL-18-positive tissues. In situ hybridization showed that, in contrast to the adrenal cortex, in spleen and duodenum IL-18 is primarily localized to immune cells. In duodenum, IL-18 mRNA was also detectable in epithelial cells. Northern blot demonstrated that while the adrenal gland synthesized IL-18 mRNA of 1.1 kb, spleen and duodenum produced a 0.9-kb transcript. RT-PCR, sequencing, Western blot, primer extension, and rapid amplification of cDNA end analysis demonstrated that the three tissues synthesize IL-18 mRNAs containing the same coding region and produce the same IL-18 peptide, but differ in the length of their 5'-untranslated region, indicating tissue-specific usage of the promoter region. Finally, in contrast to the adrenal gland, adrenocorticotropic hormone treatment did not increase the levels of IL-18 mRNA in spleen and duodenum. These results demonstrate tissue-specific expression and promoter usage of IL-18 gene and suggest that the adrenal cortex and not immune cells may be the source of IL-18 during stress.

Modulation of IL-18 production in the adrenal cortex following acute ACTH or chronic corticosterone treatment.

Interleukin (IL)-18 is a proinflammatory cytokine and a stimulator of cell-mediated immune responses. We have previously reported that acute stress stimulates the production of IL-18 mRNA in the glucocorticoid (GC)-producing cells of the adrenal cortex. In order to investigate the mechanisms governing the expression of IL-18 in the adrenal cortex, the effects of acute ACTH or chronic corticosterone treatment on the levels of IL-18 mRNA and protein were examined by in situ hybridization and Northern and Western blot assays. Adult male Sprague-Dawley rats received a subcutaneous injection of ACTH or subcutaneous implantation of slow-release corticosterone pellets followed by an injection of saline or ACTH. After 4 h, ACTH induced a 4-fold increase in IL-18 mRNA levels and elevated the content of pro-IL-18 peptide. Six days of chronic corticosterone treatment did not alter the basal levels of IL-18 mRNA and reduced those of pro-IL-18. Finally, ACTH treatment of animals under the corticosterone regimen induced a 2-fold increase in IL-18 mRNA and elevated the levels of the pro-IL-18 protein. The levels of the precursor, p45, and the active subunit p10 peptides of the IL-18-processing enzyme, IL-1beta-converting enzyme (ICE), showed no substantial differences in all the conditions tested. IL-1beta was not detected under these experimental conditions. These data demonstrate that the production of IL-18 in the adrenal cortex is stimulated by ACTH treatment and is not inhibited by the direct action of corticosterone. In contrast to the anti-inflammatory action of GCs, IL-18 may have an immunostimulatory role during acute stress.

Morphometry of the head of the caudate nucleus in patients with velocardiofacial syndrome (del 22q11.2).

UNLABELLED: Velocardiofacial syndrome (VCFS) is a chromosomal anomaly syndrome characterized by multiple congenital malformations, including cleft palate and cardiac anomalies. Many patients have attention-deficit (hyperactivity) disorders (AD[H]D) in childhood and schizophrenia in adulthood. We reviewed cranial magnetic resonance imaging (MRI) scans with particular attention to the head of the caudate nucleus and found that in control subjects, the head of the caudate was larger on the left than on the right, whereas VCFS patients showed a reversed right > left pattern or no significant asymmetry. A similar right > left asymmetry or a lack of this asymmetry has been reported in patients with AD(H)D. CONCLUSIONS: These results suggest a common pathophysiological mechanism of behavioural and cognitive problems in patients with AD(H)D and those with VCFS.

Psychiatric inpatients and chromosome deletions within 22q11.2.

Velocardiofacial syndrome (VCFS) is a congenital disorder characterised by multiple dysmorphisms, cleft palate, cardiac anomalies, and learning disabilities due to a microdeletion of chromosome 22q11.2. Although VCFS is often associated with psychiatric symptoms, its prevalence among psychiatric patients is unknown. A total of 326 patients admitted in September and October 1997 to a Japanese psychiatric hospital were screened for the clinical features of VCFS. Twelve patients with minor facial dysmorphia were identified; chromosomal analysis with fluorescent in situ hybridisation (FISH) was performed in six patients who, further assessment suggested, were most likely to have VCFS. Chromosome 22q11.2 deletion was identified in a 41 year old woman who had symptoms of schizophrenia but no major dysmorphia, such as cardiovascular anomalies and cleft palate. Her behavioural and neuropsychological profiles were similar to those previously reported in VCFS. She was hemizygous for the FISH probe N25 (GDB locus D22S75) and also for probes N72H9 (D22S181), sc11.1a, C443 (D22S941), sc4.1 (D22S134), sc11.1b, N19B3 (D22S264), N122B5 (D22S934), and N77F7 (D22S939). The size of the deletion was about 3 Mb. Our patient had only some features of VCFS including a square nasal root, hypernasal speech, and hypoparathyroidism. She did, however, have the common larger deletion of type A. This finding suggests that psychiatric symptoms in VCFS can occur without major developmental symptoms such as cardiovascular anomalies and cleft palate. Additional patients with schizophrenia may have subtle features of VCFS which are unrecognised on routine medical examinations.

Effect of immobilization on solubility of soleus and gastrocnemius muscle collagen: -biochemical studies on collagen from soleus and gastrocnemius muscles of rat.

We have been studying mainly the changes in collagen fiber solubility with respect to its influence on immobilization of the soleus muscle and Achilles tendon of rats. We decided to investigate also the change of the collagen fiber solubility in the gastrocnemius muscle which, like the soleus muscle, is assumed to influence the range of motion of the ankle joints. The purpose of this study, therefore, was to investigate the effects of a 7-week immobilization on the solubility of the gastrocnemius muscle and soleus muscle collagen fiber of rats. The results were that in a 7-week immobilization period, hydroxyproline concentration in tissue was increased and salt and pepsin soluble collagen was decreased in both the soleus and the gastrocnemius muscles. The results suggest an increase in the collagen concentration in tissue and an increase in the intermolecular cross-link with a stronger molecular structure. As far as the amount of collagen and solubility were concerned, the immobilization had a similar influence on the collagen fiber in both muscles.

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.

Pharmacological properties of the CO2/H+-sensitive area in the ventral medullary surface assessed by the effects of chemical stimulation on respiration.

We recently discovered that CO2/H+-sensitive neurons in the ventral medullary surface (VMS) are immunoreactive to glutamate, glutamic acid decarboxylase (GAD), calcineurin and cAMP. We then tested the hypothesis that glutamate, GABA, calcineurin and cAMP affect the activity of CO2/H+-sensitive neurons in the VMS. Using male Wistar rats anesthetized with urethane and pentobarbital, we checked for changes in relative tidal volume (VT) and respiratory frequency (f) in response to injecting the VMS with a variety of test agents dissolved in mock CSF. Respiratory changes occurred immediately and were dose-dependent. (1) 200-1600 pmol Glutamate increased VT but decreased f. The glutamate effect was never abolished by concomitant injection of AP5, a NMDA receptor antagonist, but was abolished by CNQX, an AMPA receptor antagonist, indicating predominance of AMPA receptors in the CO2/H+-sensitive neurons in the VMS. (2) 200-1600 pmol GABA decreased both VT and f. The GABA effect was never abolished by concomitant injection of saclofen, a GABA(B) receptor antagonist, but was abolished by bicuculline, a GABA(A) receptor antagonist, indicating predominance of GABA(A) receptors in the CO2/H+-sensitive neurons in the VMS. (3) 4-32 microg Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase 2B, and 200-1600 pmol FK506, selective inhibitor of calcineurin, had no effect on respiration when they were applied extracellularly, but 400-3200 pmol BAPTA-AM, an intracellular Ca2+-chelating agent, decreased both VT and f, indicating involvement of intracellular Ca2+ in the excitatory mechanisms of respiration. (4) 100-800 pmol IBMX, an enhancer of intracellular cAMP, decreased both VT and f, indicating involvement of cAMP in the inhibitory mechanisms of respiration. These results indicate that the CO2/H+-sensitive neurons in the VMS contain glutamate and/or GABA in cytoplasma, possess AMPA and/or GABA(A) receptors on surface of plasma membrane, and compose the internal circuit, and that their activities are regulated by Ca2+ and cAMP.

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.

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.

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.