Jari Koistinaho

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 ∼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.

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 heat shock hsp70 mRNA and HSP70 kDa protein in neurons in the ‘penumbra’ following focal cerebral ischemia in the rat

Induction of hsp70 heat shock protein (HSP70) and hsp70 mRNA was examined using adjacent sections in the same rat brain following permanent middle cerebral artery (MCA) occlusions, hsp70 mRNA was induced within 4 h of MCA occlusion and persisted for at least 24 h. Cellular resolution autoradiographs suggested that hsp70 mRNA was induced primarily in neurons in the periphery of ischemia both outside and inside of the infarction, with small amounts of hsp70 mRNA being induced in the core of the infarction. HSP70 protein was localized in neurons outside the infarction and in endothelial cells within the infarction at 24 h but not at 4 h following permanent MCA occlusions. It is proposed that the penumbra, one of the areas that can be rescued by pharmacological agents, can be defined anatomically as the volume of tissue outside the area of infarction in which HSP70 protein is expressed primarily in neurons.

Phencyclidine induction of the hsp70 stress gene in injured pyramidal neurons is mediated via multiple receptors and voltage gated calcium channels

Non-competitive N-methyl-D-aspartate receptor antagonists, including phencyclidine, ketamine, and MK801, produce vacuoles and induce the hsp 70 stress gene in layer III pyramidal neurons of the rat cingulate cortex. This study shows that phencyclidine (50 mg/kg) induces hsp 70 messenger RNA and HSP70 stress protein primarily in pyramidal neurons in posterior cingulate and retrosplenial cortex, neocortex, insular cortex, piriform cortex, hippocampus, and in the basal nuclei of the amygdala. Several neurotransmitter receptor antagonists inhibited induction of HSP70 produced by phencyclidine (50 mg/kg): haloperidol (ED50 = 0.8 mg/kg), clozapine (ED50 = 1 mg/kg), valium (ED50 = 1 mg/kg), SCH 23390 (ED50 = 7 mg/kg) and muscimol (ED50 = 3 mg/kg). Baclofen had no effect. Nifedipine blocked the induction of HSP70 produced by phencyclidine in some regions (cingulate, neocortex, insular cortex) but only partially blocked HSP70 induction in other regions (piriform cortex, amygdala). These results suggest that phencyclidine injuries pyramidal neurons via dopamine D1, D2, D4, sigma and other receptors. Several factors appear to contribute to this unusual multi-receptor mediated injury. (1) Phencyclidine blocks N-methyl-D-aspartate receptors on GABAergic interneurons resulting in decreased inhibition of pyramidal neurons. This may help to explain why multiple excitatory receptors mediate the injury and why GABAA agonists decrease the injury produced by phencyclidine. (2) Phencyclidine blockade of an amine transporter helps explain why dopamine receptor antagonists ameliorate injury. (3) Phencyclidine depolarizes neurons and produces high, potentially damaging intracellular calcium levels probably by blocking K+ channels that may be linked to sigma receptors. Since nifedipine prevents injury in cingulate, insula, and neocortex, it appears that calcium entry through L-type voltage gated calcium channels plays a role in the pyramidal neuronal injury produced by phencyclidine in these regions. There are similarities between the cingulate neurons injured by phencyclidine and circuits recently hypothesized to explain receptor changes in cingulate gyrus of schizophrenic patients. The present and previous studies also provide approaches for decreasing the clinical side effects of N-methyl-D-aspartate receptor antagonists to facilitate their possible use in the treatment of ischemia and other disorders.

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 local 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. This prolonged expression of hsp70 mRNA in cortex cannot be accounted for by changes of CBF in Tg mice during ischemia and reperfusion.

Light-induced c-fos expression in amacrine cells in the rabbit retina

Retinal neurons that express the immediate early gene c-fos after light exposure were characterized by neurotransmitter content using histochemical and immunocytochemical staining. In Northern blots the amount of c-fos mRNA peaked at 30 min, but remained detectable 60 min following light stimulation. Fos proteins were seen in the inner nuclear and ganglion cell layers, and the staining was most intense two and three hours after beginning the light exposure. In the ganglion cell layer 30-40% of Fos-immunoreactive cells were cholinergic displaced amacrine cells and 3-5% were ganglion cells. In the inner nuclear layer 24% of Fos-immunoreactive cells were Type I and 7% Type II NADPH-diaphorase-reactive (nitric oxide synthase) amacrine cells, 11% were tyrosine hydroxylase-containing cells, and 10-15% cholinergic amacrine cells. No Fos immunoreactivity was seen in serotoninergic, somatostatin- or VIP-immunoreactive cells, bipolar, horizontal or photoreceptor cells. Nicotine, kainic acid, NMDA and SCH 38393, a dopamine D1 receptor agonist, induced Fos immunostaining in the inner nuclear and ganglion cell layers, but administration of the corresponding receptor blockers mecamylamine, kynuretic acid, MK-801, haloperidol and SCH 23990 did not prevent light-induced Fos expression.

Localization of protein kinase C subspecies in the rabbit retina

The localization of PKC subspecies alpha, beta, gamma, epsilon and zeta was studied immunocytochemically in the rabbit retina. Conventional, Ca(2+)-sensitive PKC subtypes alpha, beta, gamma were all localized in different neuronal populations. The zeta-subspecies, which does not require Ca2+ for activation, was colocalized with PKC-alpha. PKC-epsilon, which is independent of Ca2+ and DAG, was colocalized with PKC-beta. Some populations of neurons, including cone bipolar cells, contained none of the PKC-subspecies studied. These results imply a cellular segregation of different signaling pathways in mammalian retina.

Induction of NGFI-A mRNA following middle cerebral artery occlusion in rats: In situ hybridization study

Middle cerebral artery (MCA) occlusion in halothane-anesthetized rats induced the zinc finger gene, NGFI-A, in brain. In situ hybridization studies showed that NGFI-A was induced throughout all of the cortex following MCA occlusion. By 24 h after MCA occlusion there was little expression of NGFI-A mRNA in the core of the MCA infarct, but the mRNA was still induced in all of cortex outside the infarct. MCA occlusion also induced this gene in subcortical structures: ipsilateral medial striatum; most of thalamus including medial and lateral geniculate nuclei; substantia nigra; and hippocampus at 4 h of MCA occlusion which generally disappeared by 24 h of MCA occlusion. Most of these structures, except for the striatum, are not supplied by the MCA. These data show that changes in brain gene expression can occur in many regions remote from an infarction.

VIP-induced stellation and immediate early gene expression in astrocytes: effects of dexamethasone

To investigate the actions of glucocorticoids (GCs) on astrocyte functions, interactions of dexamethasone and immediate early genes (IEGs) were studied in cell cultures of rat cerebral cortical astrocytes. Vasoactive intestinal peptide (VIP) induces rapid c-fos mRNA expression and morphological changes (stellation) in cultured astrocytes. Dexamethasone pretreatment decreases this ligand-induced stellation without affecting levels of c-fos mRNA. Moreover VIP does not induce c-jun, jun-B, and NGFI-A mRNA, suggesting that these IEGs may not mediate ligand-induced stellation. The expression of c-fos, c-jun, jun-B, and NGFI-A mRNA are rapidly induced in cultured astrocytes after treatment with phorbol ester, epidermal growth factor, and basic fibroblast growth factor. Dexamethasone pretreatment has no effect on the IEG response induced by any of these agents, suggesting that GCs may not have direct effects on the promoter of these IEGs in cortical astrocytes.

Age-related increase inc-fos expression in the sympathetic neurons of the rat superior cervical ganglion

Abstract C- fos expression was studied immunocytochemically in sympathetic neurons of the rat superior cervical ganglion (SCG). Fos-like immunoreactivity was confined to the principal neurons of the ganglion and was exclusively localized within their nuclei. In 2-month-old rats, the immunoreactivity was detected in 1.2% of the principal neurons with a density of 4.95 Fos-positive cells/mm 2 of ganglion area. This proportion increased with age and reached a value which was 6.5-fold higher in the 26-month-old rats than that in the young adult. A density of 24.5 Fos-positive cells/mm 2 of ganglion area was seen in the 26-month-old animals. The age-enhancedc- fos expression suggests that Fos may be involved in regulation of the genetic events associated with the adaptive changes in neuronal activity of the sympathetic ganglion during aging.