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Cardiocirculatory arrest is the most common clinical cause of global cerebral ischemia. We studied neuronal cell damage and neuronal stress response after cardiocirculatory arrest and subsequent cardiopulmonary resuscitation in rats. The temporospatial cellular reactions were assessed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining of DNA fragments, in situ hybridization (heat shock protein hsp70; immediate early genes c-fos and c-jun), and immunocytochemical (HSP70; and myeloperoxidase, specific marker of polymorphonuclear leukocytes [PMNL]) techniques. Cardiac arrest of 10 minutes' duration was induced in mechanically ventilated male Sprague-Dawley rats anesthetized with nitrous oxide and halothane. After cardiopulmonary resuscitation, animals were allowed to reperfuse spontaneously for 6 hours, 24 hours, 3 days, and 7 days (n = 6 per group). Five sham-operated animals were controls. The TUNEL staining revealed an early onset degeneration in the thalamic reticular nucleus (TRN) at 6 hours that peaked at 3 days. In contrast, degeneration was delayed in the hippocampal CA1 sector, showing an onset at 3 days and a further increase in the number of TUNEL-positive cells at 7 days. A minor portion of TUNEL-positive nuclei in the CA1 sector showed condensed chromatin and apoptotic bodies, whereas all nuclei in the TRN revealed more diffuse staining. After 6 hours of reperfusion, levels of mRNA for hsp70 and c-jun were elevated in circumscribed areas of cortex, in all hippocampal areas, and in most nuclei of thalamus, but not in the TRN. After 24 hours, a strong expression of mRNA for hsp70 and c-jun could be observed in the second layer of the cortex and in hippocampal CA1 sector; hsp70 also was observed in hippocampal CA3 sector. Some animals showed expression of hsp70 and c-jun in the dentate gyrus. After 3 days, hsp70 and c-jun were detected mainly in the CA1 sector of hippocampus. At 7 days, mRNA for both returned to control values. Therefore, delayed cell degeneration in the CA1 sector corresponds to a prolonged expression of hsp70 and c-jun in this area. In situ hybridization studies for c-fos revealed a strong signal in CA3 and dentate gyrus and a less prominent signal in TRN at 6 hours. At 24 hours, CA4 and amygdalae were positive, whereas at 3 and 7 days, the signal reached control levels; no prolonged or secondary expression was observed in the CA1 sector. Immunohistochemical study confirmed translation of HSP70 in various areas corresponding to the detection of mRNA, including the CA1 sector. The number of PMNL increased significantly at 6 hours and 7 days after cardiac arrest; PMNL were distributed disseminately and were not regionally associated with neuronal cell damage. The current data support the view that CA1 neurons might undergo an apoptosis-associated death after cardiac arrest, but PMNL are not directly involved in this process. The marked differences in the time course and the characteristics of TUNEL staining and the neuronal stress response in CA1 sector and TRN point to different mechanisms of neuronal injury in the two selectively vulnerable areas.

A reproducible model of thread occlusion of the middle cerebral artery (MCA) was established in C57 Black/6J mice by matching the diameter of the thread to the weight of the animals. For this purpose, threads of different diameter (80 to 260 microns) were inserted into the MCA of animals of different weights (18 to 33 g), and the success of vascular occlusion was evaluated by imaging the ischemic territory on serial brain sections with carbon black. Successful occlusion of the MCA resulted in a linear relationship between body weight and thread diameter (r = 0.46, P < 0.01), allowing precise selection of the appropriate thread size. Laser-Doppler measurements of CBF, neurological scoring, and 2,3,5-triphenyltetrazolium chloride staining confirmed that matching of animal weight and suture diameter produced consistent cerebral infarction. Three hours after MCA occlusion, imaging of ATP, tissue pH, and cerebral protein synthesis allowed differentiation between the central infarct core, in which ATP was depleted, and a peripheral penumbra with reduced protein synthesis and tissue acidosis but preserved ATP content. Perfusion deficits and ischemic tissue alterations could also be detected by perfusion- and diffusion-weighted magnetic resonance imaging, demonstrating the feasibility of dynamic evaluations of infarct evolution. The use of multiparametric imaging techniques in this improved MCA occlusion model opens the way for advanced pathophysiological studies of stroke in gene-manipulated animals.

The heterogeneity of c-fos and hsp72 mRNA expression during focal ischemia was studied in mice by combining in situ hybridization with metabolic imaging. Focal ischemia was produced by middle cerebral artery occlusion for 3 h. The infarct core and the penumbra were differentiated by regional ATP and cerebral protein synthesis (CPS) imaging. hsp72 mRNA expression was restricted to the ischemic penumbra, as defined by the dissociation between preserved ATP and suppressed CPS. c-fos mRNA was expressed not only in the penumbra but also in the peri-ischemic normal brain tissue in which both ATP and CPS were preserved. These data demonstrate a highly selective differential expression of immediate-early and stress-related genes in the peri-infarct surrounding which is explained by different mechanisms of gene induction.

In view of the protective effect of brain derived neurotrophic factor (BDNF) against metabolic/excitotoxic insults in vitro, we investigated whether BDNF could limit infarct size after permanent occlusion of the middle cerebral artery in rat (MCAO). BDNF was delivered into the territory of the middle cerebral artery via an osmotic mini-pump (1 microg/h). Infusion of BDNF was started shortly after MCAO, and 24 h later brains were removed for infarct volume determination. Intraoperative and postoperative measurements of physiological variables revealed no differences among vehicle-treated and BDNF-infused animals. However, we found a 33% reduction in total infarct volume in BDNF-treated animals (p<0.05), and a 37% reduction in cortical infarct volume (p<0.05).

Moderate changes in body temperature can influence the outcome of cerebral ischemic insults and the effect of drugs. Body temperature was measured continuously for 24 hours in rats subjected to permanent occlusion of the middle cerebral artery (MCA) by either coagulation or thread insertion, and the results correlated with the histology of the hypothalamus. The body temperature did not change after MCA occlusion by coagulation and the hypothalamus was intact in all rats. In contrast, the body temperature rapidly increased from about 38 degrees C to more than 39.5 degrees C after MCA occlusion using intraluminal thread, and hyperthermia continued for at least 6 hours in all rats. Histological evaluation revealed neuronal damage in the preoptic area of the hypothalamus in all rats undergoing thread occlusion. Long duration hyperthermia must be prevented after permanent MCA occlusion when the intraluminal thread occlusion model is used in chronic experiments.

Following transient forebrain ischemia selective and delayed neuronal degeneration occurs in the CA1 sector of the hippocampus. It is presently unclear whether this cell death is related to programmed cell death (PCD), which occurs in neurons during development of the CNS. Recently, the expression of various genes, such as c-fos, c-jun mkp-1, cyclin D1, and hsp70 was found to be associated with PCD in model systems. We and others have described that these genes are also upregulated in the hippocampus following ischemia. Most notably, c-fos, c-jun, and hsp70 are expressed specifically in CA1 neurons at survival times shortly preceding cell degeneration in rat models of global ischemia. In addition, the gene products could be detected by immunohistochemical methods, despite a general impairment of protein synthesis. These finding are especially relevant, since recent report suggests a functional role for Fos family proteins and c-jun in PCD in neurons of the superior cervical ganglion. These results could be indicative for the occurrence of a PCD-related program in CA1 neurons ad corroborate several other lines of evidences, such as occurrence of DNA fragmentation. Clearly, further studies are necessary to elucidate the functional role of the gene inductions following ischemia in vivo.

Microglial cell activation is a rapidly occurring cellular response to cerebral ischaemia. Microglia proliferate, are recruited to the site of lesion, upregulate the expression of several surface molecules including major histocompatibility complex class I and II antigens, complement receptor and the amyloid precursor protein (APP) as well as newly expressed cytokines, e.g. interleukin-1 and transforming growth factor beta 1. The ischaemia-induced production of APP may contribute to amyloid deposition in the aged brain under conditions of hypofusion. Ultrastructurally, microglia transform into phagocytes removing necrotic neurons but still respecting the integrity of eventually surviving neurons even in the close vicinity of necrotic neurons. Microglial activation starts within a few minutes after ischaemia and thus precedes the morphologically detectable neuronal damage. It additionally involves a transient generalized response within the first 24 hours post-ischaemia even at sites without eventual neuronal cell death. In functional terms, the microglial reaction appears to be a double-edged sword in ischaemia. Activated microglia may exert a cytotoxic effector function by releasing reactive oxygen species, nitric oxide, proteinases or inflammatory cytokines. All of these cytotoxic compounds may cause bystander damage following ischaemia. Pharmacological suppression of microglial activation after ischaemia has accordingly attenuated the extent of cell death and tissue damage. However, activated microglia support tissue repair by secreting factors such as transforming growth factor beta 1 which may limit tissue damage as well as suppress astroglial scar formation. In line with ultrastructural observations microglial activation in ischaemia is a strictly controlled event. By secreting cytokines and growth factors activated microglia most likely serve seemingly opposed functions in ischaemia, i.e. maintenance as well as removal of injured neurons. Post-ischaemic pharmacological modulation of microglial intervention in the cascade of events that lead to neuronal necrosis may help to improve the structural and functional outcome following CNS ischaemia.

The beta-amyloid precursor protein (APP) bears characteristics of an acute-phase protein and therefore is likely to be involved in the glial response to brain injury. In the brain, APP is rapidly synthesized by activated glial cells in response to comparatively mild neuronal lesions, e.g., a remote peripheral nerve injury. Perfusion deficits in the brain result largely in neuronal necrosis and are a common condition in elderly patients. This neuronal necrosis is accompanied by a pronounced reaction of astrocytes and microglia, which can also be observed in animal models. We have therefore studied in the rat, immunocytochemically, the induction of APP after 30 min of global ischemia caused by four-vessel occlusion. The postischemic brain injuries were examined at survival times from 12 h to 7 days. From day 3 onward, APP immunoreactivity was strongly induced in the CA1 and CA4 regions of the rat dorsal hippocampus as well as in the dorsolateral striatum. In these areas, the majority of APP-immunoreactive cells were reactive glial fibrillary acidic protein (GFAP)-positive astrocytes, as shown by double-immunofluorescence labeling for GFAP and APP. Additionally, small ramified cells, most likely activated microglia, expressed APP immunoreactivity. In contrast, in the parietal cortex, APP immunoreactivity occurred focally in clusters of activated microglia rather than in astrocytes, as demonstrated by double-immunofluorescence labeling for APP and the microglia-binding lectin Griffonia simplicifolia isolectin B4. In conclusion, following global ischemia, APP is induced in reactive glial cells with spatial differences in the distribution pattern of APP induction in astrocytes and microglia.

PAC-1 mRNA has previously been found only in activated T-cells in vitro and in vivo. The gene encodes a dual specificity protein phosphatase that regulates MAP kinase activity. Here, I describe that PAC-1 mRNA is induced also in neurons in the rat brain following 30 min of forebrain ischemia. At 6, 12 and 24 h after ischemia, PAC-1 mRNA was found most prominently in hippocampal cells which are resistant to 30 min of forebrain ischemia, but not in the selectively vulnerable CA1 sector. At later time points and in control animals no PAC-1 mRNA could be detected in any brain region. The protein-tyrosine/threonine phosphatase PAC-1, therefore, may be involved in adaptational responses of hippocampal cells resistant to ischemic injury.

The temporospatial expression pattern of four immediate early genes (IEGs) (c-fos, c-jun, junB, NGFI-B) following 30 min of global ischemia was investigated in rat brains by in situ hybridization and immunohistochemistry (c-fos). All examined IEG mRNAs, as well as Fos-like immunoreactivity, increased transiently in vulnerable and resistant brain regions following ischemia, but the induction profiles were distinct. Ischemia caused a post-ischemic early-onset, transient c-fos induction in wide-spread regions, as well as a late-onset induction restricted to vulnerable regions. Late-onset c-fos induction was observed in the CA1 region and the ventral thalamus but not in the striatum or neocortex, where neurons degenerate at a quicker pace. After recirculation, c-jun mRNA appeared to be initially coinduced with c-fos mRNA, but c-jun mRNA levels remained elevated or increased in various regions, including all vulnerable regions, when c-fos mRNA had already declined to near basal levels. Compared to c-fos and c-jun, junB induction was less pronounced and confined largely to the dentate gyrus. NGFI-B mRNA increased moderately and only in brain regions exhibiting the most dramatic c-fos increases and with similar kinetics. The differential activation of the investigated IEGs suggests that rather complex long-term adaptive processes may be initiated at the genomic level after global ischemia. The present findings provide further evidence that the activation of IEGs forms part of the brain's metabolic response to ischemia, but no simple correlation appears to exist between the induction of the investigated IEGs and the phenomenon of selective vulnerability.

Expression of sulfated glycoprotein-2 (SGP-2) mRNA was studied by in situ hybridization in rat brains submitted to transient forebrain ischemia of 30 min. Induction of this multifunctional protein has been previously observed following diverse types of brain lesions, and an involvement in programmed cell death and synaptic remodelling has been proposed. Ischemia was produced by four-vessel occlusion and followed by various recirculation times ranging from 15 min to 7 days. Up to 6 h after ischemia SGP-2 mRNA did not change in any brain region. After 12 h recirculation, SGP-2 mRNA induction was observed in the stratum lacunosum moleculare of CA1 sector of hippocampus. This induction peaked at 3 days recirculation and then declined. From 24 h recirculation onward, induction also occurred in patchy areas of the cortex, and after 7 days recirculation in the ventral thalamus and in a corona around lesioned parts of the striatum. No induction occurred at any recirculation time in pyramidal neurons of hippocampus or other neuronal populations that are damaged by ischemia. The combination of in situ hybridization with GFAP immunohistochemistry revealed that SGP-2 mRNA was mainly induced in reactive actrocytes. This excludes a direct involvement in ischemic neuronal death and supports the possible participation in the post-lesional reorganization of the tissue.

Transforming growth factor-beta 1 (TGF-beta 1) and interleukin-1 beta mRNA expression were studied in rat brains after 30 min of global ischemia by in situ hybridization. Ischemia was produced by four-vessel occlusion followed by different recirculation times ranging between 15 min and 7 days. TGF-beta 1 mRNA could first be detected 3 days after ischemia in the hippocampus, in layers II/III of cortex, in the striatum and in parts of the ventral thalamus. At 7 days after recirculation a prominent increase in TGF-beta 1 mRNA was observed in the CA1 sector of the hippocampus. Induction of interleukin-1 beta mRNA, however, was less marked and limited to the rostral striatum 3 and 7 days after ischemia. TGF-beta 1 expression 7 days after ischemia correlated well with the histological localization of regions where neuronal degeneration and subsequent astrocytic and microglial activation had occurred. In adjacent brain sections, the distribution of TGF-beta 1 mRNA after 7 days closely resembled that of the immunostaining pattern of activated microglia, indicating that at this time point TGF-beta 1 mRNA was mainly produced by microglial cells. The late induction of TGF-beta 1 mRNA after ischemia points to an involvement in the persistent glial response rather than the initial glial activation. The differential pattern of interleukin-1 beta mRNA induction indicates regional variations of cytokine production after ischemic brain lesions.

Differentiated primary cortical neuronal cultures of rat were exposed for 5 min to 0.1 and 1.0 mM glutamate. In cultures maintained in serum-free medium after glutamate exposure, ribosomes completely disaggregated and neurons died within 24 h already after 0.1 mM glutamate. Addition of 5% horse serum to the culture medium prevented both ribosomal disaggregation and neuronal death even after exposure to 1.0 mM glutamate. Glutamate toxicity in vitro requires removal of serum-associated growth factors from the incubation medium and, therefore, may not be representative for neuronal vulnerability in vivo.

The primary structure of thioredoxin f from spinach chloroplasts was determined by standard amino acid sequencing and furthermore by sequencing the corresponding nuclear genome region. The protein, with a calculated molecular mass of 12,564 Da and a molar absorption coefficient at 280 nm of 17,700 M-1 cm-1, consists of 113 residues and exhibits 24% residue identities with spinach chloroplast thioredoxin mb or Escherichia coli thioredoxin. A monospecific antibody elicited against thioredoxin f has been used to select recombinant phage from spinach cDNA libraries in lambda gt11. The inserts of positive clones were sequenced. They code for a polypeptide of 190 amino acids, composed of the thioredoxin f sequence (113 residues) and an upstream element (77 residues) which most probably constitutes the N-terminal transit peptide that directs the polypeptide into chloroplasts. In vitro transcription and translation of this construct generates a polypeptide of approximately 21 kDa, which is imported by isolated spinach chloroplasts and processed to the mature 12.5-kDa protein.