HX600, a synthetic agonist for RXR-Nurr1 heterodimer complex, prevents ischemia-induced neuronal damage.

Ischemic stroke is amongst the leading causes of death and disabilities. The available treatments are suitable for only a fraction of patients and thus novel therapies are urgently needed. Blockage of one of the cerebral arteries leads to massive and persisting inflammatory reaction contributing to the nearby neuronal damage. Targeting the detrimental pathways of neuroinflammation has been suggested to be beneficial in conditions of ischemic stroke. Nuclear receptor 4A-family (NR4A) member Nurr1 has been shown to be a potent modulator of harmful inflammatory reactions, yet the role of Nurr1 in cerebral stroke remains unknown. Here we show for the first time that an agonist for the dimeric transcription factor Nurr1/retinoid X receptor (RXR), HX600, reduces microglia expressed proinflammatory mediators and prevents inflammation induced neuronal death in in vitro co-culture model of neurons and microglia. Importantly, HX600 was protective in a mouse model of permanent middle cerebral artery occlusion and alleviated the stroke induced motor deficits. Along with the anti-inflammatory capacity of HX600 in vitro, treatment of ischemic mice with HX600 reduced ischemia induced Iba-1, p38 and TREM2 immunoreactivities, protected endogenous microglia from ischemia induced death and prevented leukocyte infiltration. These anti-inflammatory functions were associated with reduced levels of brain lysophosphatidylcholines (lysoPCs) and acylcarnitines, metabolites related to proinflammatory events. These data demonstrate that HX600 driven Nurr1 activation is beneficial in ischemic stroke and propose that targeting Nurr1 is a novel candidate for conditions involving neuroinflammatory component.

Age-related decrease in stimulated glutamate release and vesicular glutamate transporters in APP/PS1 transgenic and wild-type mice.

We assessed baseline and KCl-stimulated glutamate release by using microdialysis in freely moving young adult (7 months) and middle-aged (17 months) transgenic mice carrying mutated human amyloid precursor protein and presenilin genes (APdE9 mice) and their wild-type littermates. In addition, we assessed the age-related development of amyloid pathology and spatial memory impaired in the water maze and changes in glutamate transporters. APdE9 mice showed gradual spatial memory impairment between 6 and 15 months of age. The stimulated glutamate release declined very robustly in 17-month-old APdE9 mice as compared to 7-month-old APdE9 mice. This age-dependent decrease in stimulated glutamate release was also evident in wild-type mice, although it was not as robust as in APdE9 mice. When compared to individual baselines, all aged wild-type mice showed 25% or greater increase in glutamate release upon KCl stimulation, but none of the aged APdE9 mice. There was an age-dependent decline in VGLUT1 levels, but not in the levels of VGLUT2, GLT-1 or synaptophysin. Astrocyte activation as measured by glial acidic fibrillary protein was increased in middle-aged APdE9 mice. Blunted pre-synaptic glutamate response may contribute to memory deficit in middle-aged APdE9 mice.

Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia.

Minocycline, a semisynthetic tetracycline derivative, protects brain against global and focal ischemia in rodents. We examined whether minocycline reduces excitotoxicity in primary neuronal cultures. Minocycline (0.02 microm) significantly increased neuronal survival in mixed spinal cord (SC) cultures treated with 500 microm glutamate or 100 microm kainate for 24 hr. Treatment with these excitotoxins induced a dose-dependent proliferation of microglia that was associated with increased release of interleukin-1beta (IL-1beta) and was followed by increased lactate dehydrogenase (LDH) release. The excitotoxicity was enhanced when microglial cells were cultured on top of SC cultures. Minocycline prevented excitotoxin-induced microglial proliferation and the increased release of nitric oxide (NO) metabolites and IL-1beta. Excitotoxins induced microglial proliferation and increased the release of NO metabolites and IL-1beta also in pure microglia cultures, and these responses were inhibited by minocycline. In both SC and pure microglia cultures, excitotoxins activated p38 mitogen-activated protein kinase (p38 MAPK) exclusively in microglia. Minocycline inhibited p38 MAPK activation in SC cultures, and treatment with SB203580, a p38 MAPK inhibitor, but not with PD98059, a p44/42 MAPK inhibitor, increased neuronal survival. In pure microglia cultures, glutamate induced transient activation of p38 MAPK, and this was inhibited by minocycline. These findings indicate that the proliferation and activation of microglia contributes to excitotoxicity, which is inhibited by minocycline, an antibiotic used in severe human infections.

Fos-like immunoreactivity in the rat hypothalamic-pituitary axis after immobilization stress.

The effect of immobilization stress on the expression of the protooncogene c-fos in the rat pituitary and hypothalamus was investigated immunohistochemically using different polyclonal antibodies raised against the c-fos protein (Fos). After a 4 h immobilization, Fos-like immunoreactivity (Fos-LI) increased substantially in the parvocellular part of the paraventricular nucleus and in the intermediate and anterior lobe of the pituitary. The majority of the Fos-immunoreactive cells in the pituitary contained corticotropin, which was demonstrated by immunohistochemical double-staining. Since the paraventricular nucleus contains a large number of glucocorticoid receptor immunoreactive cells, the effect of a synthetic glucocorticoid, dexamethasone, on the induction of Fos-LI was studied. Dexamethasone treatment before immobilization considerably reduced the stress-induced expression of Fos-LI in the anterior and intermediate lobe of the pituitary but did not alter the induction of Fos-LI in the paraventricular nucleus. The present results demonstrate that immobilization stress induces Fos-LI both in the hypothalamus and in the pituitary, suggesting that Fos may be involved in regulating the synthesis of different mediators of stress response, such as CRF- and POMC-derived peptides. Apparently glucocorticoids do not directly repress c-fos expression, since dexamethasone did not affect the induction of Fos-LI in the paraventricular nucleus. The reduction of stress-induced Fos-LI in the pituitary by dexamethasone is possibly due to the diminished release of CRF factor from the paraventricular neurons.

Induction of protein kinase Cdelta subspecies in neurons and microglia after transient global brain ischemia.

The delayed death of CA1 neurons after global brain ischemia is associated with induction of apoptosis genes and is inhibited by protein synthesis inhibitors, suggesting that the degeneration of CA1 pyramidal neurons is an active process that requires new gene expression. The transient global ischemia model has been extensively used to identify enzymes and other proteins underlying delayed neuronal cell death. The expression of protein kinase C (PKC) subspecies after 20 minutes of global brain ischemia produced by a four-vessel occlusion model in the rat was studied. From the multiple PKC subspecies studied, only PKCdelta mRNA was significantly up-regulated in CA1 pyramidal neurons at 24 hours and in activated microglia at 3 to at least 7 days after ischemia. The induction of PKCdelta mRNA was also found in the cortex at 8 hours and 3 days after ischemia. This cortical but not hippocampal induction was regulated by an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptor antagonist, 6-nitro-7-sulfamobenzo[f]quinoxaline-2,3-dione, and glucocorticoids. An N-methyl-D-aspartate receptor antagonist, MK-801, was without effect on the induction of PKCdelta subspecies. The selective and prolonged induction of the PKCdelta mRNA and protein first in CA1 pyramidal neurons and at a later stage in activated microglia suggests that the PKCdelta isozyme may take part in regulation of the delayed death of CA1 neurons after transient global brain ischemia.

Induction of c-fos, junB, c-jun, and hsp70 mRNA in cortex, thalamus, basal ganglia, and hippocampus following middle cerebral artery occlusion.

Middle cerebral artery (MCA) occlusion in halothane-anesthetized rats induced c-fos, junB, and c-jun immediate early gene mRNAs and hsp70 heat shock gene mRNA in brain. In situ hybridization studies showed that c-fos and junB were induced throughout all of the cortex at 1 and 4 h following MCA occlusion. hsp70 was induced in the core and margins of the MCA ischemia. By 24 h, there was little expression of c-fos, junB, c-jun, and hsp70 in the core of the MCA infarct; there was modest induction of hsp70 at the margins of the infarct; and there was diffuse induction of c-fos, junB, and c-jun in all of the cortex outside the infarct. MCA occlusion also induced these genes in subcortical structures. c-fos, junB, and hsp70 were induced in ipsilateral medial striatum, most of thalamus including medial and lateral geniculate nuclei, substantia nigra, and hippocampus. Most of these structures, except for the striatum, are not supplied by the MCA. These data show that changes in gene expression can occur in regions remote from an infarction.

Induction of 70-kDa heat shock protein and hsp70 mRNA following transient focal cerebral ischemia in the rat.

Induction of the 70-kDa heat shock protein (HSP70) was demonstrated immunocytochemically in adult rats 4 h to 7 days following temporary middle cerebral artery (MCA) occlusions lasting 30, 60, or 90 min. Maximal HSP70 induction occurred approximately 24 h following ischemia. Thirty minutes of ischemia induced HSP70 in neurons throughout the cortex in the MCA distribution, whereas 90 min of ischemia induced HSP70 in neurons in the penumbra. HSP70 protein was induced in endothelial cells in infarcted neocortex following 60-90 min of MCA occlusion, and HSP70 was induced in endothelial cells in infarcted regions of lateral striatum following 30-90 min of MCA occlusion. hsp70 mRNA was induced in the MCA distribution in cortex and to a lesser extent in striatum at 2 h to 3 days following 60 min of ischemia. It is proposed that brief ischemia induces hsp70 mRNA and HSP70 protein in the cells most vulnerable to ischemia--the neurons. HSP70 protein is not induced in most neurons and glia following 60-90 min of ischemia in areas destined to infarct, whereas it is induced in vascular endothelial cells.

Prolonged expression of hsp70 mRNA following transient focal cerebral ischemia in transgenic mice overexpressing CuZn-superoxide dismutase.

The distribution of heat shock protein hsp70 mRNA after 10 min of middle cerebral artery (MCA) occlusion was investigated through in situ hybridization in transgenic (Tg) mice overexpressing CuZn-superoxide dismutase (CuZn-SOD) and in control nontransgenic (nTg) littermates. In the ischemic cortex of nTg mice, hsp70 mRNA was detected 1 h after reperfusion and was observed for up to 6 h. In Tg mice, however, it was still detectable within the cortex even at 24 h. In the caudate putamen, hsp70 mRNA appeared at 1 h and was present for up to 6 h in both nTg and Tg mice. Although hsp70 mRNA was detected in the thalamus only at 1 h in nTg mice, it was observed for up to 6 h in Tg mice. Similarly, hsp70 mRNA was detected in the hippocampus of nTg mice only at 1 h, whereas it was detected in Tg mice at 1 h and continued up to 24 h, with high intensity in the CA1 subfield. Despite the significant amounts of hsp70 mRNA in both Tg and nTg mice following ischemia, there was no observable neuronal necrosis (as assessed using hematoxylin and eosin staining) for up to 7 days. Cortical cerebral blood flow (CBF), measured by laser-Doppler flowmetry, did not differ between nTg and Tg mice during ischemia and reperfusion, despite exhibiting hyperemia following hypoperfusion. These results suggest that oxidative stress affects the expression of hsp70 following temporary focal ischemia. An alteration in oxidation stress, which resulted from reduced levels of superoxide radicals in the presence of the CuZn-SOD transgenes, may permit the prolonged expression of hsp70.(ABSTRACT TRUNCATED AT 250 WORDS)

Sympathetic neurons outlive the original host after transplantation to the rat submandibular gland.

Sympathetic ganglion tissue of aged (36 months) Wistar rats was allotransplanted into the submandibular gland (SMG) of young (3 months) animals to study whether sympathetic neurons can outlive the original host. The viability of the transplants was evaluated one year postgrafting, using the formaldehyde-induced fluorescence technique (FIF) for histochemical demonstration of catecholamines, tyrosine hydroxylase (TH) immunohistochemistry, and morphometry. One year after transplantation, grafted superior cervical ganglion (SCG) cells demonstrated catecholamine fluorescence and tyrosine hydroxylase immunoreactivity. The transplants consisted of groups of sympathetic neurons dispersed in a fibrous matrix. After long postgrafting time, the sympathetic neurons of aged rats showed several signs of enhanced degeneration; increased autofluorescent lipopigment, decreased neuronal density and reduced catecholamine fluorescence. The mean diameter of the transplanted aged neurons was significantly decreased. The histograms of grouped diameter values showed a shift to smaller cells in ganglion transplants. A subpopulation of small and medium-sized grafted neurons sent out fluorescent fibers, which were located in a fibrous scar area but did not extend into submandibular host tissue. The results indicate that a long postgrafting time induced degeneration which is comparable to normal aging changes in grafted very old neurons. Thus, aged sympathetic neurons maintain plasticity to survive as transplants, and under favourable conditions these neurons outlive the original host.

Molecular cloning and characterization of the rat inducible nitric oxide synthase (iNOS) gene.

We have cloned and characterized the rat inducible nitric oxide synthase (iNOS) gene. It spans approx. 36kb and is divided into 27 exons and 26 introns. The distribution and length of exons are similar to those in the human iNOS gene. In the 5' flanking regulatory region of the rat iNOS gene, there are a number of putative transcription factor binding sites (>20), many of them probably indispensable for the gene's nuclear factor kappaB (NFkappaB)-dependent induction, but also many which may have a role in its NFkappaB-independent induction pathway. These include cyclic adenosine 3', 5'-monophosphate (cAMP) response elements (CRE), hypoxia responsive element (HRE) and GATA-core elements. Rat models are powerful tools in studies of neurological diseases. Because iNOS is most likely responsible for the harmful consequences of nitric oxide (NO) in general, the cloned rat iNOS gene will further reveal the mechanisms of iNOS inducibility in different cell types during development and disease, including brain diseases, and to promote studies of pharmacological intervention in cases where extensive NO production plays a critical role.

Genomic structure and chromosomal localization of the rat protein kinase Cdelta-gene.

Protein kinase Cdelta (PKCdelta) is a widely expressed calcium-independent PKC isozyme that is induced at mRNA and protein levels upon stimulation of different cellular pathways. We found the rat PKCdelta gene to consist of 19 exons and to span approximately 29 kb. The exon-intron junctions follow the GT/AG rule. The 5' untranslated region is nearly 12 kb in length, and the transcription initiation site is surrounded by CG-rich sequences. The 5' flanking region contains putative binding sites for activator protein 1 (AP-1), nuclear factor kappa B (NFkappaB), stimulatory protein-1 (Sp-1) and nerve growth factor induced-C (NGFI-C) transcription factors. The PKCdelta gene is localized at the rat chromosome 19p14. The cloned gene will help to elucidate the role of PKCdelta in growth, differentiation and death of mammalian cells.

Protein kinase C-beta-like immunoreactivity and phosphorylation dependent immunoreactivity of neurofilaments in developing, adult and aged rat peripheral neurons.

The localizations of protein kinase C-beta-like immunoreactivity (PKC-beta-LI) and phosphorylated neurofilament-like immunoreactivity (PNF-LI) were studied in developing (E12-P28), adult (3-month-old) and aged (15- and 30-month-old) rat dorsal root (DRG) and superior cervical (SCG) ganglia. A close correlation was found between the localization of PKC-beta-LI and PNF-LI in all age groups. PKC-beta-LI was already present in the rat DRG at the time of the appearance of PNF-LI (E12) and after E15 both PKC-beta-and PNF-LI were found in the same subpopulation of neuronal perikarya. Aging had no effect on the distribution or intensity of either PKC-beta or PNF-LI. In the SCG neurons no association was observed between immunoreactivity and accumulation of lipopigments, but in the DRG neurons, lipopigment accumulation was seen in PKC-beta and PNF-immunoreactive large neurons. The results suggest that PKC-beta may regulate the time of appearance and phosphorylation state of some forms of phosphorylated neurofilaments during development. The results also show that no major change occurs in the amount or distribution of PNFs during normal aging.

Light and electron microscopic features of peripheral ganglion cells cultured from young and aged Wistar rats.

Neurons of the dorsal root ganglion (DRG) and sympathetic superior cervical ganglion (SCG) were cultured as explants from young adult (3 months old) and aged (28 months old) Wistar rats. Both aged DRG and SCG neurons showed delayed neurite outgrowth compared to young adult neurons. Young and some aged cultured neurons had an ultrastructure similar to that found in normal uncultured cells, but most of the aged cultured neurons displayed a heavy accumulation of homogenous lipid-like inclusions in addition to classic age pigments. Occasionally, large neurofilament aggregates were seen in aged DRG neurons. They were sometimes localized perinuclearly, resembling neurofibrillary tangles. The results show that even very old peripheral neurons survive in culture. As aged cultured neurons show degenerative changes not observed in young adult neurons, it is suggested that cultured peripheral neurons of different ages could provide useful means for neuronal aging studies.

The effect of bleaching on the lipopigments in the human sympathetic neurons.

The effect of exposure to H2O2 on the lipopigments of the human sympathetic neurons was investigated by fluorescence and electron microscopy. In intact ganglia two subtypes of the lipopigment were seen: the yellow fluorescent pigment (emission maximum at 510 nm), which under the electron microscope had two components (vacuoles and osmiophilic matrix) and the orange fluorescent pigment (emission maximum at 535 nm), which in electron microscopy showed a third, highly osmiophilic component. Bleaching did not change the emission spectrum of the yellow pigment, while that of orange pigment shifted from 535 nm to 485 nm. Under electron microscopy the highly osmiophilic component disappeared in bleached orange pigment bodies. The characteristics of orange autofluorescent lipopigment are similar to those described for neuromelanin in the substantia nigra. The results suggest that neuromelanin accumulates in aging sympathetic neurons.

Effect of sialectomy on the superior cervical ganglion sympathetic neurons in young adult and aged mice.

The dependence of superior cervical ganglion (SCG) adrenergic neurons on their target organ submandibular salivary gland containing high concentrations of nerve growth factor was studied in adult and aged male mice. The submandibular salivary glands were removed (sialectomy) either uni- or bilaterally, and the SCG were studied by fluorescence microscopy and histochemically. Catecholamine fluorescence and tyrosine hydroxylase immunoreactivity decreased after sialectomy, suggesting reduced noradrenaline production. Neuronal density was lower in the aged controls than in the young controls. In both age groups, sialectomy reduced the density of catecholamine-producing neurons. In the mouse SCG, there was remarkable heterogeneity in the size of neuronal somata. In aged control mice there was a greater number of large-size neurons than in young adult control mice. Six weeks postoperatively, no large catecholamine-producing neurons could be observed in the ganglia. Yellow autofluorescent lipopigments accumulated with age in the adrenergic neurons. Sialectomy increased the accumulation of lipopigments in both young and aged neurons. Sialectomy resulted in (a) reduced catecholamine fluorescence, (b) reduced tyrosine hydroxylase immunoreactivity, (c) reduced number of catecholamine neurons, (d) increased autofluorescent lipopigment. Ageing resulted in (a) reduced number of neurons, (b) increased ratio of large to small neurons, (c) increased autofluorescent lipopigment. Alterations after sialectomy were more detrimental in the aged ganglia than in the young adult ganglia. The discontinuation of the retrograde supply of nerve growth factor may contribute to these alterations.

Effect of vitamin E and selenium supplement on the aging peripheral neurons of the male Sprague-Dawley rat.

The effect of life-long diets containing different concentrations of selenium and vitamin E on the age pigment accumulation in the rat superior cervical ganglion, vagal ganglion and dorsal root ganglion, was studied using microspectrofluorometry. All types of ganglia showed unchanged amounts of age pigments at low or high concentrations of selenium, whereas dietary concentration of vitamin E regulated age pigment content in the dorsal root ganglion, but not in the superior cervical and vagal ganglion. Vitamin E deficiency induced a three-fold increase in age pigment content in dorsal root ganglion at 8 months of age, whereas high vitamin E concentration was associated with a lesser amount of pigments at 18 months of age. Emission spectra of age pigment recorded from the dorsal root ganglion and vagal ganglion were different from that from the superior cervical ganglion, but were independent of dietary concentrations of selenium or vitamin E. The results suggest that exogenous antioxidants may play a more crucial role in lipid peroxidation and accumulation of age pigment in dorsal root ganglion than in autonomic ganglia.

Microspectrofluorometric quantitation of autofluorescent lipopigment in the human sympathetic ganglia.

The age-related changes in lipopigment autofluorescence were studied by microspectrofluorometry in three different types of human neurons: the sympathetic neurons of the stellate and superior mesenteric ganglion and pyramidal neurons of the frontal cortex. The age-related increase in lipopigment autofluorescence was more rapid in stellate ganglion but similar linear increases were found also in superior mesenteric ganglion and frontal cortex. There was an age-related shift in the autofluorescence from yellow to orange in the ganglia. This may be due to the accumulation of neuromelanin in noradrenergic neurons. Lipopigments were identified in sympathetic neurons at the age of 4 months and all neurons carried pigment granules after the age of 64 years. It is concluded that lipopigment autofluorescence is a useful marker for cellular ageing in both the peripheral and the central nervous system.

Adrenergic innervation of the tibial and vagus nerves in rats of different ages.

Adrenergic nerve fibers innervating blood vessels in the epi-perineurium and endoneurium of the tibial and vagus nerves of male Fischer-344 rats of different ages were examined using formaldehyde-induced fluorescence technique and fluorescence microscopy. Between 6 and 24 months of age, no significant difference in the mean density of perivascular innervation in the epi-perineurium of the nerves was found, whereas between 24 months and 30 months the density decreased in both nerve types. The intensity of noradrenaline fluorescence of nerve fibers also was decreased in the oldest age group. In the endoneurium, the density of adrenergic nerve fibers was reduced by 57% at 24 months, and by more than 98% at 30 months as compared with 18 months. Age-related differences were similar in the tibial and vagus nerves. The results suggest that adrenergic neuronal control of the microcirculation in the rat tibial and vagus nerves is lost during aging as it is in other organs of the cardiovascular system.

Histochemically demonstrable catecholamines in the sympathetic nervous system of trisomic 16 and normal fetal mice.

The formaldehyde-induced fluorescence (FIF) and micro-spectrofluorometric techniques were used to study catecholamines in the sympathetic nervous system of normal and trisomy 16 fetal mice with a gestation age of 15 days, an animal model for human trisomy 21 (Down's syndrome). FIF intensity in the stellate sympathetic ganglion of trisomic embryos did not differ from that of controls, whereas in the adrenal medulla the FIF intensity was 38% less in trisomic than in control embryos. When adrenal medullary cells from embryos with a gestational age of 15 days were maintained in culture for 7-10 days, a difference in FIF intensity between groups was still evident. The rate of noradrenaline uptake in cultured adrenal medullary cells also was significantly less in trisomic than in control fetal mice. The results suggest that the development of adrenal medulla is slowed in trisomic 16 mice and that uptake of noradrenaline by trisomic adrenal medullary cells is impaired.

Blood-nerve and blood-brain barrier permeabilities and nerve vascular space in Fischer-344 rats of different ages.

The permeability-surface area product (PA) of [3H]- or [14C]sucrose at the blood-nerve barrier (BNB) of the sciatic nerve; and at the blood-brain barrier (BBB), were determined in Fischer-344 rats at 3, 11 and 31 months of age. PA was determined by using an in vivo i.v. bolus injection of radiotracer with two-time point graphic and quantitative autoradiographic methods. Vascular space and water content of the tibial nerve of these rats also were determined using quantitative morphometry and dry and wet weight ratios, respectively. There was no significant difference between mean PA(BNB) in any age group [(PA(BNB) at 3 months = 1.2 +/- 0.1 (mean +/- S.E.), at 11 months = 1.8 +/- 0.3; and at 31 months = 1.4 +/- 0.2 x 10(5) ml/s . g wet wt; n = 5-8 rats], nor any difference in PA(BBB). The mean ratio (%) of surface area of endoneurial blood vessels/nerve cross-section of the tibial nerve also did not differ between any group [3 months: 16 +/- 2 vessels; mean surface area ratios = 2.20 +/- 0.10%, n = 5; 11 months: 22 +/- 3 vessels and 2.48 +/- 0.21%, n = 5; 11 months: 22 +/- 3 vessels and 2.48 +/- 0.21%, n = 5; and at 31 months: 26 +/- 1 vessels and 2.40 +/- 0.23%, n = 4). The mean nerve water in rats at 31 months was 64.8 +/- 1.1% wet wt and did not differ from that at 11 months (66.0 +/- 0.6% wet wt) or at 3 months (65.1 +/- 1.0% wet wt) (n = 5-8 nerves). Our results indicate that BBB and BNB integrities are not altered in senescent Fischer-344 rats.