Adoption of a Urologic Oncology Perioperative Surgical Home is Associated with Decreased Total Length of Stay: A Pilot Study.

INTRODUCTION: Urologists partnered with anesthesiologists to implement a model of perioperative and postoperative care known as the multidisciplinary perioperative surgical home in order to improve the quality and efficiency of care. We describe early outcomes associated with implementation of the perioperative surgical home. METHODS: Retrospective chart review was performed of patients at a single institution undergoing radical prostatectomy, radical cystectomy, partial nephrectomy and radical nephrectomy from January 2014 to March 2016. Outcomes measured were length of stay and 30-day reoperation, readmission, unexpected intensive care unit admission and mortality rates. Statistical analysis was performed using the independent samples Mann-Whitney U test and Fisher exact test with p <0.05 considered significant. Univariate and multivariate analyses were performed to determine whether implementation of the perioperative surgical home was associated with improved outcomes. RESULTS: Length of hospital stay decreased from 4.79 to 3.19 days and 30-day complication rate decreased from 15.3% to 5.7% after implementation of the perioperative surgical home (p <0.01 for both). There was no change in the 30-day readmission rate. On multivariate analysis surgery occurring after perioperative surgical home implementation was associated with decreased length of stay (p = 0.008). The direct cost savings resulting from this length of stay reduction totaled $1,245,585 for the study period. CONCLUSIONS: The adoption of a perioperative surgical home is associated with a significantly decreased postoperative hospital stay and 30-day complication rate for urologic oncology cases.

Expression of miR-1294 is downregulated and predicts a poor prognosis in gastric cancer.

OBJECTIVE: MicroRNAs (miRNAs) play critical roles in regulating tumor development and progression. The aim of the study is to investigate the clinical significance of miR-1294 expression in gastric cancer (GC). PATIENTS AND METHODS: The expression of miR-1294 in 82 cases of GC tissues and adjacent normal tissues was determined using quantitative Real Time-PCR (qRT-PCR) analyses. Survival plot was calculated using the Kaplan-Meier methods and log-rank test from the date of operation to the time of death or last follow-up date. The association between miR-1294 expression and clinical categorical data was analyzed using the chi-squared test. Moreover, Univariate and multivariate Cox analysis were performed to assess the risk factors of GC prognosis. RESULTS: We showed that miR-1294 expression was significantly downregulated in GC tissues compared to adjacent normal tissues. The low expression of miR-1294 in patients with GC was correlated with clinicopathological parameters including larger tumor size, lymph node metastasis, and distant metastasis. Kaplan-Meier survival analysis showed that GC patients with lower miR-1294 expression exhibited a shorter disease-free survival (DFS) and overall survival (OS) time compared to those patients with higher miR-1294 expression. Multivariate Cox analysis showed that lower miR-1294 expression, tumor size, lymph node metastasis, and distant metastasis were identified as independent risk factors of GC prognosis. CONCLUSIONS: Our results provided evidence that miR-1294 expression was significantly downregulated in GC and may serve as a predictor of GC prognosis.

Irreversible aggregation of protein synthesis machinery after focal brain ischemia.

Focal brain ischemia leads to a slow type of neuronal death in the penumbra that starts several hours after ischemia and continues to mature for days. During this maturation period, blood flow, cellular ATP and ionic homeostasis are gradually recovered in the penumbral region. In striking contrast, protein synthesis is irreversibly inhibited. This study used a rat focal brain ischemia model to investigate whether or not irreversible translational inhibition is due to abnormal aggregation of translational complex components, i.e. the ribosomes and their associated nascent polypeptides, protein synthesis initiation factors and co-translational chaperones. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons, but clumped into large abnormal aggregates in penumbral neurons subjected to 2 h of focal ischemia followed by 4 h of reperfusion. The abnormal ribosomal protein aggregation lasted until the onset of delayed neuronal death at 24-48 h of reperfusion after ischemia. Biochemical study further suggested that translational complex components, including small ribosomal subunit protein 6 (S6), large subunit protein 28 (L28), eukaryotic initiation factors 2alpha, 4E and 3eta, and co-translational chaperone heat-shock cognate protein 70 (HSC70) and co-chaperone Hdj1, were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational complex components were also highly ubiquitinated. This study clearly demonstrates that focal ischemia leads to irreversible aggregation of protein synthesis machinery that contributes to neuronal death after focal brain ischemia.

Co-translational protein aggregation after transient cerebral ischemia.

Transient cerebral ischemia leads to irreversible translational inhibition which has been considered as a hallmark of delayed neuronal death after ischemia. This study utilized a rat transient cerebral ischemia model to investigate whether irreversible translational inhibition is due to abnormal aggregation of translational complex, i.e. the ribosomes and their associated nascent polypeptides, initiation factors, translational chaperones and degradation enzymes after ischemia. Translational complex aggregation was studied by electron microscopy, as well as by biochemical analyses. A duration of 15 or 20 min of cerebral ischemia induced severe translational complex aggregation starting from 30 min of reperfusion and lasting until the onset of delayed neuronal death at 48 h of reperfusion. Under electron microscopy, most rosette-shaped polyribosomes were relatively evenly distributed in the cytoplasm of sham-operated control neurons. After ischemia, most ribosomes were clumped into large abnormal aggregates in neurons destined to die. Translational complex components consisting of small ribosomal subunit protein 6, large subunit protein 28, eukaryotic initiation factor-3eta, co-translational chaperone heat shock cognate protein 70 and co-chaperone HSP40-Hdj1, as well as co-translational ubiquitin ligase c-terminus of hsp70-interacting protein were all irreversibly clumped into large abnormal protein aggregates after ischemia. Translational components were also highly ubiquitinated. To our knowledge, irreversible aggregation of translational components has not been reported after brain ischemia. This study clearly indicates that ischemia damages co-translational chaperone and degradation machinery, resulting in irreversible destruction of protein synthesis machinery by protein aggregation after ischemia.

Ischemic preconditioning prevents protein aggregation after transient cerebral ischemia.

Transient cerebral ischemia leads to protein aggregation mainly in neurons destined to undergo delayed neuronal death after ischemia. This study utilized a rat transient cerebral ischemia model to investigate whether ischemic preconditioning is able to alleviate neuronal protein aggregation, thereby protecting neurons from ischemic neuronal damage. Ischemic preconditioning was introduced by a sublethal 3 min period of ischemia followed by 48 h of recovery. Brains from rats with either ischemic preconditioning or sham-surgery were then subjected to a subsequent 7 min period of ischemia followed by 30 min, 4, 24, 48 and 72 h of reperfusion. Protein aggregation and neuronal death were studied by electron and confocal microscopy, as well as by biochemical analyses. Seven minutes of cerebral ischemia alone induced severe protein aggregation after 4 h of reperfusion mainly in CA1 neurons destined to undergo delayed neuronal death (which took place after 72 h of reperfusion). Ischemic preconditioning reduced significantly protein aggregation and virtually eliminated neuronal death in CA1 neurons. Biochemical analyses revealed that ischemic preconditioning decreased accumulation of ubiquitin-conjugated proteins (ubi-proteins) and reduced free ubiquitin depletion after brain ischemia. Furthermore, ischemic preconditioning also reduced redistribution of heat shock cognate protein 70 and Hdj1 from cytosolic fraction to protein aggregate-containing fraction after brain ischemia. These results suggest that ischemic preconditioning decreases protein aggregation after brain ischemia.

Pathogenesis of hippocampal neuronal death after hypoxia-ischemia changes during brain development.

Transient hypoxia-ischemia (HI) leads to delayed neuronal death in both mature and immature neurons but the underlying mechanisms are not fully understood. To understand whether the pathogenesis of HI-induced neuronal death is different between mature and immature neurons, we used a rat HI model at postnatal days 7 (P7), 15 (P15), 26 (P26) and 60 (P60) in order to investigate ultrastructural changes and active caspase-3 distribution in HI-injured neurons as a function of developmental age. In P7 pups, despite more than 95% of HI-injured neurons highly expressing active caspase-3, most of these active caspase-3-positive neurons revealed mixed features of apoptosis and necrosis (a chimera type) under electron microscopy (EM). Classical apoptosis was observed only in small populations of HI-injured P7 neurons. Furthermore, in rats older than P7, most HI-injured neurons displayed features of necrotic cell death under EM and, concomitantly, active caspase-3-positive neurons after HI declined dramatically. Classical apoptosis after HI was rarely found in neurons older than P15. In P60 rats, virtually all HI-injured neurons showed the shrinkage necrotic morphology under EM and were negative for active caspase-3. These results strongly suggest that pathogenesis of HI-induced neuronal death is shifting from apoptosis to necrosis during brain development.

Protein aggregation after focal brain ischemia and reperfusion.

Two hours of transient focal brain ischemia causes acute neuronal death in the striatal core region and a somewhat more delayed type of neuronal death in neocortex. The objective of the current study was to investigate protein aggregation and neuronal death after focal brain ischemia in rats. Brain ischemia was induced by 2 hours of middle cerebral artery occlusion. Protein aggregation was analyzed by electron microscopy, laser-scanning confocal microscopy, and Western blotting. Two hours of focal brain ischemia induced protein aggregation in ischemic neocortical neurons at 1 hour of reperfusion, and protein aggregation persisted until neuronal death at 24 hours of reperfusion. Protein aggregates were found in the neuronal soma, dendrites, and axons, and they were associated with intracellular membranous structures during the postischemic phase. High-resolution confocal microscopy showed that clumped protein aggregates surrounding nuclei and along dendrites were formed after brain ischemia. On Western blots, ubiquitinated proteins (ubi-proteins) were dramatically increased in neocortical tissues in the postischemic phase. The ubi-proteins were Triton-insoluble, indicating that they might be irreversibly aggregated. The formation of ubi-protein aggregates after ischemia correlated well with the observed decrease in free ubiquitin and neuronal death. The authors concluded that proteins are severely damaged and aggregated in neurons after focal ischemia. The authors propose that protein damage or aggregation may contribute to ischemic neuronal death.

Early release of cytochrome C and activation of caspase-3 in hyperglycemic rats subjected to transient forebrain ischemia.

The mechanisms underlying the aggravating effect of hyperglycemia on brain damage are still elusive. The present study was designed to test our hypothesis that hyperglycemia-mediated damage is caused by mitochondrial dysfunction with mitochondrial release of cytochrome c (cyt c) to the cytoplasm, which leads to activation of caspase-3, the executioner of cell death. We induced 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of recirculation in sham, normoglycemic and hyperglycemic rats. Release of cyt c was observed in the neocortex and CA3 in hyperglycemic rats after only 0.5 h of reperfusion, when no obvious neuronal damage was observed. The release of cyt c persisted after 1 and 3 h of reperfusion. Activation of caspase-3 was observed after 1 and 3 h of recovery in hyperglycemic animals. No cyt c release or caspase-3 activation was observed in sham-operated controls while a mild increase of cyt c was observed in normoglycemic ischemic animals after 1 and 3 h of reperfusion. The findings that there is caspase activation and cyt c relocation support a notion that the biochemical changes that constitute programmed cell death occur after ischemia and contribute, at least in part, to hyperglycemia-aggravated ischemic neuronal death.

Protein ubiquitination in rat brain following hypoglycemic coma.

Hypoglycemic coma of 30 min duration selectively damages CA1 pyramidal neurons and the crest of dentate gyrus (DG) granule cells in hippocampus. Here, we show by high-resolution confocal microscopy and biochemical analysis that 30 min of hypoglycemic coma induces the ubiquitination and aggregation of several proteins in rat brain tissues. Protein ubiquitination and aggregation occurred in the CA1 and DG regions as early as the end of 30 min of hypoglycemic coma and lasted until neuronal death in the late recovery period after hypoglycemia. In comparison, the neurons surviving hypoglycemia were less affected. On western blots, ubiquitinated proteins (ubi-proteins) were present mainly in Triton-insoluble pellets, indicating that they are irreversibly aggregated. We conclude that proteins are ubiquitinated and aggregated in neurons after hypoglycemia prior to their death. We hypothesize that protein ubiquitination and aggregation may contribute to neuronal damage after hypoglycemia.

Phosphorylation of extracellular signal-regulated kinase after transient cerebral ischemia in hyperglycemic rats.

The present study was undertaken to investigate whether extracellular signal-regulated kinase (ERK) was involved in mediating hyperglycemia-exaggerated cerebral ischemic damage. Phosphorylation of ERK 1/2 was studied by immunocytochemistry and by Western blot analyses. Rats were subjected to 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of reperfusion under normoglycemic and hyperglycemic conditions. The results showed that in normoglycemic animals, moderate phosphorylation of ERK 1/2 was transiently induced after 0.5 h of recovery in cingulate cortex and in dentate gyrus, returning to control values thereafter. In hyperglycemic animals, phosphorylation of ERK 1/2 was markedly increased in the cingulate cortex and dentate gyrus after 0.5 h of recovery, the increases being sustained for at least 3 h after reperfusion. Hyperglycemia also induced phosphorylation of ERK 1/2 in the hippocampal CA3 sector but not in the CA1 area. Thus, the distribution of phospho-ERK 1/2 coincides with hyperglycemia-recruited damage structures. The results suggest that hyperglycemia may influence the outcome of an ischemic insult by modulating signal transduction pathways involving ERK 1/2.

Alterations of Akt1 (PKBalpha) and p70(S6K) in transient focal ischemia.

The serine-threonine kinase Akt1 promotes cell survival through inhibition of apoptosis. One of the potential downstream targets of Akt1 is p70 S6 kinase, p70(S6K), an enzyme implicated in the regulation of protein synthesis. In this study, we investigated the changes in total and phosphorylated levels of Akt1 and p70(S6K) during transient focal ischemia. Male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by 1, 4, and 24 h of reperfusion. The expression of total and phosphorylated forms of Akt1 and p70(S6K) were examined by Western blot analysis. Phosphorylation of Akt1 on Ser473 transiently increased at 1 and 4 h of reperfusion, whereas phosphorylation of Akt1 on Thr308 was reduced during reperfusion. The levels of total Akt1 remained unchanged at 1 and 4 h of reperfusion, but decreased significantly at 24 h of reperfusion. Phosphorylation of p70(S6K) on Thr389 decreased at 1, 4, and 24 h of reperfusion, while the levels of total p70(S6K) protein remained unchanged at 1 and 4 h of reperfusion but decreased at 24 h of reperfusion. The results show that cell survival pathways, such as Akt1 and p70(S6K) signaling, are suppressed after transient focal ischemia, which may contribute to the development of neuronal cell death after an ischemic insult.

Alterations of hippocampal postsynaptic densities following transient ischemia.

A transient interruption in cerebral blood flow can lead to delayed neuronal death in certain vulnerable cell populations several days after blood flow is restored. Among the most vulnerable cell populations in the forebrain are hippocampal CA1 pyramidal neurons, which die between 48-72 h after the ischemic insult. Neurons in the dentate gyrus and area CA3 are relatively resistant, and will recover from the same insult. Uncovering the factors that render some neuronal populations vulnerable to transient ischemia is key to understanding mechanisms leading to cell death and to developing therapeutic interventions. By applying selective staining and three-dimensional (3D) imaging with electron tomography, we uncovered dramatic structural modifications in postsynaptic densities in the postischemic brain. Postsynaptic densities in the postischemic brain appeared both thicker and less condensed than those from sham-operated controls. Although the class of synapse could not be determined with the methods used, most are likely to be glutamatergic synapses onto dendritic spines, because the majority of synapses in the region examined belong to this class. Further analysis using electron tomography to examine the 3D structure of postsynaptic densities revealed degenerative changes, as evidenced by an overall loosening of the normally compact structure. Synaptic modifications were particularly severe and persistent in hippocampal area CA1 compared to the dentate gyrus. These structural modifications correlate well with biochemical and physiological studies indicating that alterations in synaptic transmission occur in the postischemic brain. The combination of selective staining and 3D reconstruction provides a valuable tool for revealing aspects of synaptic morphology not apparent from standard electron microscopic evaluation.

Alteration of cyclic adenosine monophosphate response element binding protein in rat brain after hypoglycemic coma.

In the current study, the temporal and regional changes of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB) were investigated in rat brains subjected to 30 minutes of hypoglycemic coma followed by varied periods of recovery using Western blot and confocal microscopy. The total amount of CREB was not altered in any area examined after coma. The level of the phosphorylated form of CREB decreased during coma but rebounded after recovery. In the relatively resistant areas, such as the inner layers of the neocortex and the inner and outer blades of the dentate gyms (DG), phospho-CREB increased greater than the control level after 30 minutes of recovery and continued to increase up to 3 hours of recovery. In contrast, little or no increase of phospho-CREB was observed during the recovery period in the outer layers of the neocortex and at the tip of the DG, that is, regions that are selectively vulnerable to hypoglycemic insults. The current findings suggest that a neuroprotective signaling pathway may be more activated in the resistant regions than in the vulnerable ones after hypoglycemic coma.

Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation.

The involvement of caspase-3 in cell death after hypoxia-ischemia (HI) was studied during brain maturation. Unilateral HI was produced in rats at postnatal day 7 (P7), 15 (P15), 26 (P26), and 60 (P60) by a combination of left carotid artery ligation and systemic hypoxia (8% O2). Activation of caspase-3 and cell death was examined in situ by high-resolution confocal microscopy with anti-active caspase-3 antibody and propidium iodide and by biochemical analysis. The active caspase-3 positive neurons were composed of more than 90% HI damaged striatal and neocortical neurons in P7 pups, but that number was reduced to approximately 65% in striatum and 34% in the neocortex of P15 pups, and approximately 26% in striatum and 2% in neocortex of P26 rats. In P60 rats, less than 4% of the damaged neurons in striatum and less than 1% in neocortex were positive for active caspase-3. Western blot analysis demonstrated that the level of inactive caspase-3 in normal forebrain tissue gradually declined from a high level in young pups to very low levels in adult rats. Concomitantly, HI-induced active caspase-3 was reduced from a relatively high level in P7, to moderate levels in P15 and P26, to a barely detectable level in P60 rats. The authors conclude that the involvement of caspase-3 in the pathogenesis of cell death after HI declines during neuronal maturation. The authors hypothesize that caspase-3 may play a major role in cell death in immature neurons but a minor role in cell death in mature neurons after brain injury.

Differential phosphorylation at Ser473 and Thr308 of Akt-1 in rat brain following hypoglycemic coma.

We analyzed both total Akt-1 and phosphorylation of Akt-1 at residues Ser473 and Thr308 (phospho-Akt-1(Ser474) and phospho-Akt-1(Thr308), respectively) in the outer and inner layers of cortex following 30 min of hypoglycemic coma by Western blot analyses and confocal microscopy. The total amount of Akt-1 was not altered in any area examined. Phospho-Akt-1(Ser474), however, increased significantly in both layers of cortex at 0 and 30 min of recovery, but returned to control level at 3 h of recovery. In the vulnerable area (outer layer of cortex), no upregulation of phospho-Akt-1(Thr308) was observed at any time points examined. In the resistant area like inner layer of cortex, however, phospho-Akt-1(Thr308) was significantly over the control level at 3 h of recovery. Confocal microscopy result indicates that most of phospho-Akt-1(Thr308) had already moved into nucleus at 3 h of recovery. Our results suggest that Akt-1, when phosphorylated at Thr308, may play a protective role for neurons in the resistant regions of the brain.

Alteration of MAP kinase pathways after transient forebrain ischemia.

Extracellular regulated kinase (ERK) transduce growth factor signals while c-Jun NH(2)-terminal kinase (JNK) delivers stress signals into the nuclei for regulation of gene expression. These signaling pathways were studied by laser-scanning confocal microcopy and Western blot analysis using phospho-specific antibodies on rat brains that were subjected to 15 minutes transient forebrain ischemia followed by varied periods of reperfusion. Extracellular regulated kinase was activated at 30 minutes and 4 hours of reperfusion in the nuclei and dendrites of surviving dentate gyrus (DG) cells, but not in dying CA1 neurons after ischemia. Tyrosine phosphorylation of Trk kinase, an ERK upstream growth factor receptor, was elevated in the DG tissue, and to a lesser extent in the CA1 region. In addition, phosphorylation of activating transcription factor-2 (ATF-2) and c-Jun was selectively increased in CA1 dying neurons during the late period of reperfusion. These findings suggested that the Trk-ERK signaling pathway might be neuroprotective for dentate granule cells. The activation of ATF-2 and c-Jun pathways in the late period of reperfusion in CA1 dying neurons might reflect damage signals in these neurons. These results suggested that the lack of protective signals acting in concert with the presence of damage signals in CA1 neurons after ischemia might contribute to delayed neuronal death after transient forebrain ischemia.

Protein aggregation after transient cerebral ischemia.

Protein aggregates containing ubiquitinated proteins are commonly present in neurodegenerative disorders and have been considered to cause neuronal degeneration. Here, we report that transient cerebral ischemia caused severe protein aggregation in hippocampal CA1 neurons. By using ethanolic phosphotungstic acid electron microscopy (EM) and ubiquitin immunogold EM, we found that protein aggregates were accumulated in CA1 neurons destined to die 72 hr after 15 min of cerebral ischemia. Protein aggregates appeared as clumps of electron-dense materials that stained heavily for ubiquitin and were associated with various intracellular membranous structures. The protein aggregates appeared at 4 hr and progressively accumulated at 24 and 48 hr of reperfusion in CA1 dying neurons. However, they were rarely observed in dentate gyrus neurons that were resistant to ischemia. At 4 hr of reperfusion, protein aggregates were mainly associated with intracellular vesicles in the soma and dendrites, and the nuclear membrane. By 24 hr of reperfusion, the aggregates were also associated with mitochondria, the Golgi apparatus, and the dendritic plasmalemma. High-resolution confocal microscopy further demonstrated that protein aggregates containing ubiquitin were persistently and progressively accumulated in all CA1 dying neurons but not in neuronal populations that survive in this model. We conclude that proteins are severely aggregated in hippocampal neurons vulnerable to transient brain ischemia. We hypothesize that the accumulation of protein aggregates cause ischemic neuronal death.

Survival- and death-promoting events after transient cerebral ischemia: phosphorylation of Akt, release of cytochrome C and Activation of caspase-like proteases.

Release of cytochrome c (cyt c) into cytoplasm initiates caspase-mediated apoptosis, whereas activation of Akt kinase by phosphorylation at serine-473 prevents apoptosis in several cell systems. To investigate cell death and cell survival pathways, the authors studied release of cyt c, activation of caspase, and changes in Akt phosphorylation in rat brains subjected to 15 minutes of ischemia followed by varying periods of reperfusion. The authors found by electron microscopic study that a portion of mitochondria was swollen and structurally altered, whereas the cell membrane and nuclei were intact in hippocampal CA1 neurons after 36 hours of reperfusion. In some neurons, the pattern of immunostaining for cyt c changed from a punctuate pattern, likely representing mitochondria, to a more diffuse cytoplasmic localization at 36 and 48 hours of reperfusion as examined by laser-scanning confocal microscopic study. Western blot analysis showed that cyt c was increased in the cytosolic fraction in the hippocampus after 36 and 48 hours of reperfusion. Consistently, caspase-3-like activity was increased in these hippocampal samples. As demonstrated by Western blot using phosphospecific Akt antibody, phosphorylation of Akt at serine-473 in the hippocampal region was highly increased during the first 24 hours but not at 48 hours of reperfusion. The authors conclude that transient cerebral ischemia activates both cell death and cell survival pathways after ischemia. The activation of Akt during the first 24 hours conceivably may be one of the factors responsible for the delay in neuronal death after global ischemia.

Modification of postsynaptic densities after transient cerebral ischemia: a quantitative and three-dimensional ultrastructural study.

Abnormal synaptic transmission has been hypothesized to be a cause of neuronal death resulting from transient ischemia, although the mechanisms are not fully understood. Here, we present evidence that synapses are markedly modified in the hippocampus after transient cerebral ischemia. Using both conventional and high-voltage electron microscopy, we performed two- and three-dimensional analyses of synapses selectively stained with ethanolic phosphotungstic acid in the hippocampus of rats subjected to 15 min of ischemia followed by various periods of reperfusion. Postsynaptic densities (PSDs) from both area CA1 and the dentate gyrus were thicker and fluffier in postischemic hippocampus than in controls. Three-dimensional reconstructions of selectively stained PSDs created using electron tomography indicated that postsynaptic densities became more irregular and loosely configured in postischemic brains compared with those in controls. A quantitative study based on thin sections of the time course of PSD modification indicated that the increase in thickness was both greater and more long-lived in area CA1 than in dentate gyrus. Whereas the magnitude of morphological change in dentate gyrus peaked at 4 hr of reperfusion (140% of control values) and declined thereafter, changes in area CA1 persisted and increased at 24 hr of reperfusion (191% of control values). We hypothesize that the degenerative ultrastructural alteration of PSDs may produce a toxic signal such as a greater calcium influx, which is integrated from the thousands of excitatory synapses onto dendrites, and is propagated to the neuronal somata where it causes or contributes to neuronal damage during the postischemic phase.

Persistent phosphorylation of cyclic AMP responsive element-binding protein and activating transcription factor-2 transcription factors following transient cerebral ischemia in rat brain.

The transcription factors cyclic AMP responsive element-binding protein (CREB) and activating transcription factor-2 were studied in rat brains subjected to 15 min ischemia followed by varied periods of reperfusion using western blot and immunocytochemical analyses. The total amounts of both CREB and activating transcription factor-2 were not altered in the hippocampus after ischemia. In contrast, levels of the phosphorylated forms of both transcription factors decreased during ischemia but rebounded following reperfusion. The phospho-forms of CREB and activating transcription factor-2 showed regional and temporal differences in their expression. Phospho-CREB was increased relative to control levels at 30 min, and continued to increase for at least three days postischemia, mainly in dentate granule cells. The level of phospho-activating transcription factor-2 appeared to be higher in CAI pyramidal cells than in dentate granule cells after ischemia. The present findings suggest that the signaling pathways for phosphorylation of CREB may be neuroprotective for dentate cells, which are relatively resistant to ischemic insults. The increased phospho-activating transcription factor-2 may reflect increased stresses in these neurons. The more modest activation of CREB pathways in CA1 neurons may not be enough to overcome the increased stresses in these neurons, contributing to delayed neuronal death.