Marc A. Soriano

Induction of cyclooxygenase-2 mRNA and protein following transient focal ischemia in the rat brain.

Expression of cyclooxygenase-2 (cox-2) mRNA and inducible heat-shock protein-70 (hsp-70) mRNA was studied with in situ hybridization techniques at 30 min and 4 h following 1 h transient middle cerebral artery (MCA) occlusion in the rat brain. In addition, immunoreactivity for cox-2 was studied after 8 h of reperfusion. Induction of hsp-70 and cox-2 mRNA was found in the brain side ipsilateral to MCA occlusion. Hsp-70 mRNA was induced in the parietal cortex and striatum within the territory of the occluded MCA. Induction of cox-2 mRNA was particularly seen in cortical layer II in the brain side ipsilateral to MCA occlusion. At 30 min of reperfusion, areas showing cox-2 mRNA induction included the cingulate and frontal cortices located perifocally to the areas showing hsp-70 mRNA induction, and the piriform cortex. At 4 h of reperfusion, induction of cox-2 mRNA was seen within the parietal cortex. At 8 h of reperfusion, immunoreactivity for cox-2 was mainly seen in the ipsilateral cortex. These results demonstrate that transient focal ischemia induces the expression of cox-2 mRNA and protein in discrete areas of the rat brain during reperfusion, which might lead to local increases of arachidonic acid metabolism.

The heat shock stress response after brain lesions: Induction of 72 kDa heat shock protein (cell types involved, axonal transport, transcriptional regulation) and protein synthesis inhibition

The cerebral stress response is examined following a variety of pathological conditions such as focal and global ischemia, administration of excitotoxins, and hyperthermia. Expression of 72 kDa heat shock protein (Hsp70) and hsp70 mRNA, the mechanism underlying induction of hsp70 mRNA involving activation of heat shock factor 1, and inhibition of cerebral protein synthesis are different aspects of the stress response considered here. The results are compared with those in the literature on induction, transcriptional regulation, expression, and cellular location of Hsp70, with a view to getting more insight into the function of the stress response in the injured brain. The present results illustrate that Hsp70 can be expressed in cells affected at various degrees following an insult that will either survive or dic as the brain lesion develops, depending on the severity of cell injury. This indicates that, under certain circumstances, synthesized Hsp70 might be necessary but not sufficient to ensure cell survival. Other situations involve uncoupling between synthesis of hsp70 mRNA and protein, probably due to very strict protein synthesis blockade, and often result in cell loss. Cells eventually will die if protein synthesis rates do not go back to normal after a period of protein synthesis inhibition. The stress response is a dynamic event that is switched on in neural cells sensitive to a brain insult. The stress response is, however, tricky, as affected cells seem to need it, have to deal transiently with it, but eventually be able to get rid of it, in order to survive. Putative therapeutic treatments can act either selectively, potentiating the synthesis of Hsp70 protein and recovery of protein synthesis, or preventing the stress response by deadening the insult severity.

Apoptosis and c-Jun in the thalamus of the rat following cortical infarction.

Cortical infarction produces secondary neuronal damage in the thalamus. In this study we examined the thalamus of the rat following 2 h occlusion of the middle cerebral artery (MCA), and found degeneration and gliosis in the ipsilateral ventropostero-medial thalamic nucleus in those rats that showed cortical infarction 7 and 14 days after occlusion. This was accompanied by isolated cells with fragmented DNA, as revealed by in situ labelling of nuclear DNA fragmentation, and showing morphological features of apoptosis, i.e. chromatin condensation, extreme nuclear shrinkage and apoptotic bodies. In addition, cells immunoreactive for c-Jun showing morphological signs of apoptosis were observed. These results provide evidence of apoptosis in the ipsilateral thalamus following cortical infarction, and suggest that c-Jun is involved in this process.

Kainic acid-induced heat shock protein-70, mRNA and protein expression is inhibited by MK-801 in certain rat brain regions.

The regional expression of inducible 72 kDa heat shock protein (HSP‐70), HSP‐70 mRNA and the neuropathological outcome of their expression were examined in the rat brain following systemic administration of kainic acid (9 mg/kg), and also after pretreatment with the non‐competitive N‐methyl‐D‐aspartate antagonist MK‐801 (1 mg/kg). Five hours after administration of kainic acid alone, dense expression of HSP‐70 mRNA was found within the limbic system, mainly in the hippocampus, piriform and entorhinal cortices, amygdaloid complex, thalamic nuclei, subiculum and in other cortical areas in rats that had shown convulsive behaviour. At 24 h, HSP‐70 immunoreactivity was seen in most areas previously expressing HSP‐70 mRNA, except the piriform and entorhinal cortices and several ventral nuclei of the amygdaloid complex. Histopathological examination at 24 h revealed marked cell loss in these latter regions and less severe histopathological changes in other areas of the limbic system in brains of convulsive rats. No alterations were apparent in non‐convulsive rats. The percentage of rats showing convulsive behaviour with kainic acid was reduced from 74 to 4% following pretreatment with MK‐801. In addition, MK‐801 inhibited the kainic acid‐induced expression of HSP‐70 mRNA and protein in certain brain regions, notably the cortex, the pyramidal cell layer of CA1, and discrete thalamic nuclei. However, HSP‐70 mRNA induction was sustained in the pyramidal cell layer of CA3, the amygdaloid complex and the subiculum, despite the fact that none of these rats convulsed. MK‐801 prevented necrosis in all rats examined except the single rat that had shown convulsive behaviour. These results show that early regional expression of inducible HSP‐70 mRNA allows the visualization of regions affected by kainic acid and maps regions inhibited by MK‐801. In addition, the results identify brain regions putatively involved in the manifestation of limbic convulsions. Furthermore, these data illustrate that the induction of HSP‐70 mRNA is not predictive of cell death or survival.

Early 72-kDa heat shock protein induction in microglial cells following focal ischemia in the rat brain

Focal cerebral ischemia in the adult rat produces induction of 72-kDa heat shock protein (HSP-72) in neurons, glia and endothelial cells. Double antigen immunocytochemistry was carried out to find out whether microglial cells express HSP-72 following 1-h middle cerebral artery (MCA) occlusion. A monoclonal antibody against the CR3 complement receptor (OX-42) specific for microglia was used followed by a monoclonal antibody against HSP-72. Co-localization of these antibodies was seen in cells of the ipsilateral corpus callosum and striatum at 3 h following 1-h MCA occlusion, and in the ipsilateral striatal penumbra, corpus callosum and cortex at 8 h. Results demonstrate that stellate microglial cells show an early response to 1-h MCA occlusion by expressing inducible HSP-72, thus suggesting that microglial cells are sensitive to the ischemic insult.

Cortical infarct volume is dependent on the ischemic reduction of perifocal cerebral blood flow in a three-vessel intraluminal MCA occlusion/reperfusion model in the rat.

Occlusion of the middle cerebral artery (MCA) causes a reduction of cerebral blood flow (CBF), which shows a progressive decrease from the periphery to the core of the MCA territory. The severity of ischemia is dependent on the duration of the ischemic episode and degree of CBF reduction. Fixing the ischemic episode to 1 h, we have examined whether or not cortical infarct size was related to the degree of CBF reduction in a perifocal cortical area in rats. One-hour intraluminal MCA occlusion accompanied with bilateral common carotid artery (CCA) occlusion (three-vessel occlusion/reperfusion model) was carried out in Sprague-Dawley rats and CBF was monitored with laser-Doppler flowmetry in the fronto-parietal cortex, an area which is perifocal to the core of the MCA territory. Finally, infarct size was measured 7 days later and was related to the corresponding CBF decrease. Sequential ipsilateral CCA, MCA and contralateral CCA occlusions produced reductions of CBF to 96%, 52% and 33% of baseline, respectively. Cortical infarct volume was found to be dependent on the corresponding reduction of perifocal cortical CBF during the ischemic episode. These results show that the reduction of CBF in the periphery of the MCA territory during 1-h focal ischemia determines infarct size in a three-vessel occlusion/reperfusion model.

Expression of c-fos and inducible hsp-70 mRNA following a transient episode of focal ischemia that had non-lethal effects on the rat brain.

Expression of c-fos and inducible hsp-70 mRNA was studied with in situ hybridization techniques at different times following an episode of middle cerebral artery (MCA) occlusion not resulting in any apparent lethal effect on the rat brain. hsp-70 and c-fos mRNA were found in the ipsilateral striatum and adjacent cortex. In the striatum, levels of hsp-70 mRNA increased from 1 to 2 and 4 h of reperfusion, whereas levels of c-fos mRNA decreased from 1 to 4 h of reperfusion. These results demonstrate that following non-lethal focal ischemia the brain areas within the MCA territory show high c-fos and hsp-70 mRNA expression response, illustrating the concomitant induction of these mRNAs in cells that survive the ischemic insult.

Survival of parvalbumin-immunoreactive neurons in the gerbil hippocampus following transient forebrain ischemia does not depend on HSP-70 protein induction

HSP-70 was induced in the gerbil following 20 min of forebrain ischemia. The induction, as revealed with immunohistochemistry, is stronger and longer-lasting in CA3 and dentate gyrus than in CA1. Most neurons in this region, except GABAergic interneurons containing the calcium-binding protein parvalbumin, eventually cease to live as a result of delayed cell death. Double-labeling of inducible HSP-70 and parvalbumin has shown that no co-localization occurs in the hippocampus and neocortex of the gerbil in this model of transient forebrain ischemia. These results show that different thresholds of sensitivity and vulnerability exist for different subpopulations of neurons in the ischemic hippocampus, and suggest that HSP-70 protein induction is probably not essential for the survival of particular neuronal subpopulations subjected to transient ischemia.

Striatal infarction in the rat causes a transient reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra

Dopaminergic neurons of the substantia nigra pars compacta were examined in the rat brain following striatal infarction subsequent to transient focal cerebral ischemia. Rats had the middle cerebral artery occluded for 2 h or were sham-operated, and tyrosine hydroxylase immunoreactivity was evaluated by Western blot and immunohistochemistry at different times ranging from 1 to 60 days after ischemia. The number of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta was counted under the light microscope and compared to that in the contralateral side and controls. No changes of tyrosine hydroxylase immunoreactivity were detected in the ipsilateral versus the contralateral substantia nigra of sham-operated rats or 1 day after ischemia. However, a statistically significant reduction of tyrosine hydroxylase-immunoreactive cells became apparent in the ipsilateral compared with the contralateral substantia nigra at 7 and 14 days after ischemia. This reduction showed a clear recovery at 30 days after ischemia, and no signs of difference between the ipsilateral and the contralateral side were apparent by 60 days. Therefore, the reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra was only transiently seen from 1 to 2 weeks following ischemia. The observed loss of tyrosine hydroxylase was not accompanied by signs of cell death or gliosis in the ipsilateral pars compacta. The present results show a transitory reduction of tyrosine hydroxylase immunoreactivity in the ipsilateral substantia nigra pars compacta after focal ischemia and suggest that striatal infarction causes a transient deficit of dopaminergic function.

Monoclonal antibodies as markers of specific cell types and regional antigens in the freshwater planarian Dugesia (G.) tigrina

We have produced monoclonal antibodies (mAbs) against antigens of the fresh-water planarian Dugesia (G.) tigrina (Girard) using standard protocols. Labeling these mAbs with PAP (peroxidase-antiperoxidase) and indirect-immunofluorescence methods, we then determined the distribution of their antigens in the planarian. Out of 112 mAbs that showed some specificity for restricted parts of the planarian, 71 were found to be cell- or tissue-specific — among them 36 for parenchymal cells, 7 for muscle cells, 11 for epidermal cells, 8 for gastrodermis, and 7 to basement membrane. Another 41 showed different, but overlapping, regional specificities, namely to pharynx and parenchyma. So far, we have been unable to isolate specific mAbs against undifferentiated cells (neoblasts). These mAbs should be important tools in study of tissue and cell morphology, regeneration, and growth and degrowth.