Expression of c-fos, junB, c-jun, MKP-1 AND hsp72 following traumatic neocortical lesions in rats—relation to spreading depression

doi:10.1016/S0306-4522(98)00249-8

Neuroscience

Volume 88, Issue 2, January 1999, Pages 599-608

https://doi.org/10.1016/S0306-4522(98)00249-8 Get rights and content

Abstract

The effects of a traumatic neocortical lesion on c-fos, junB, c-jun, MKP-1 and hsp72 expression were examined by in situ hybridization and immunocytochemistry 1–6 h following transcranial cold injury. The direct current potential was recorded in the injury-remote cortex to evaluate the role of transient direct current shifts, i.e. spreading depressions, in gene expression. In 14 out of 21 injured rats, spreading depression-like depolarizations of the direct current potential were noticed, which were accompanied by a transient decrease in electroencephalographic activity and increase in laser Doppler flow. In seven injured animals, no spontaneous direct current deflections were seen. In animals without spreading depressions, only a short-lasting response of c-fos, junB, c-jun and MKP-1 messenger RNAs as well as c-Fos protein was bilaterally found in the piriform cortex, and—with ipsilateral dominance—the dentate gyrus and hippocampal CA3/4 fields at 1 h after lesioning. In injured animals with spreading depressions however, a strong elevation was seen in layers II–IV and VI of the injury-remote ipsilateral cerebral cortex, which persisted over as long as 6 h. Messenger RNA levels for c-fos, junB and MKP-1 were closely related to the time interval between the last direct current deflection and the end of experiment. Levels were highest shortly after transient direct current shifts, and decreased thereafter mono-exponentially with half-lives of 48, 75 and 58 min for c-fos, junB and MKP-1 messenger RNAs, respectively. In 6 h animals with spreading depressions, hsp72 messenger RNA was slightly elevated in layer II of the injury-remote cortex, but Hsp72 was not increased.

The present results demonstrate that spreading depression is the most prominent factor influencing the trauma-related gene response in the lesion-remote cortical tissue.

Section snippets

Animal experimentation

Experiments were carried out in accordance with the NIH guidelines for the care and use of laboratory animals, and approved by local government authorities. All efforts were made to minimize animal suffering. Male Sprague–Dawley rats (Harlan Winkelmann, Borchen, Germany), weighing 180–300 g, were submitted to sham surgery or subjected to cold injury of the right sensorimotor cortex, followed by 1, 3 and 6 h survival (n=7/group). Animals were anaesthetized with 0.8% halothane (30% O₂, remainder N₂

Physiological variables

Physiological variables measured in this study are summarized in Table 1. Mean values did not differ between the control group and experimental groups, suggesting no time-dependent influence on measured systemic variables after cortical lesioning.

Laser Doppler flowmetry and electrophysiological observations

Cortical d.c. potential, EEG activity and laser Doppler flow were transiently reduced during lesioning, but completely recovered within 2–5 min (Fig. 1). After lesioning, transient depolarizations of the cortical d.c. potential, followed by

Discussion

In the present report, we examined the expression of c-fos, junB, c-jun, MKP-1 and hsp72 by in situ hybridization and immunocytochemistry 1, 3 and 6 h following a traumatic lesion of the sensorimotor cortex, using an experimental setup in which transient d.c. depolarizations (so-called spreading depressions) were recorded in the injury-remote cortical tissue. Traumatic brain lesions were produced by transcranial cold injury, which gives rise to sharply demarcated lesions of the cortical tissue

Conclusions

The present results show that spreading depression is the most prominent factor influencing acute changes of gene expression following traumatic neocortical injury. Genes were assessed, which are implicated in intracellular signalling, transcription control and programmed cell death.9, 13, 18, 31, 37, 44The fact that all immediate-early genes were uniformly activated by transient d.c. depolarizations points towards the notion that spreading depression is a strong and overriding genetic

Acknowledgements

The authors gratefully acknowledge the technical assistance of Mrs M. Nelles, S. Krause and M. Fahrig. This work was supported by grant 01KO9508/0 of the “Bundesministerium für Bildung und Forschung” (BMBF).

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MKP-1 dephosphorylates the MAPK family. The expression of MKP-1 has been reported to be induced by various forms of stress including ischemia (Takano et al., 1995; Soriano et al., 2000), spreading depression (Hermann et al., 1999), and hypoxic preconditioning (Bernaudin et al., 2002). Up-regulation of the hippocampal CA1 region in a rat model of global ischemia delayed neuronal death and induced ischemic tolerance, and MKP-1 acted rather specifically as an inhibitor of JUN/p38 signaling, which is implicated in tolerance (Kawahara et al., 2004).
Mild to moderate differences in brain temperature are known to greatly affect the outcome of cerebral ischemia. The impact of brain temperature on ischemic disorders has been mainly evaluated through pathological analysis. However, no comprehensive analyses have been conducted at the gene expression level. Using a high-density oligonucleotide microarray, we screened 24 000 genes in the hippocampus under hypothermic (32 °C), normothermic (37 °C), and hyperthermic (39 °C) conditions in a rat ischemia-reperfusion model. When the ischemic group at each intra-ischemic brain temperature was compared to a sham-operated control group, genes whose expression levels changed more than three-fold with statistical significance could be detected. In our screening condition, thirty-three genes (some of them novel) were obtained after screening, and extensive functional surveys and literature reviews were subsequently performed. In the hypothermic condition, many neuroprotective factor genes were obtained, whereas cell death- and cell damage-associated genes were detected as the brain temperature increased. At all intra-ischemic brain temperatures, multiple molecular chaperone genes were obtained. The finding that intra-ischemic brain temperature affects the expression level of many genes related to neuroprotection or neurotoxicity coincides with the different pathological outcomes at different brain temperatures, demonstrating the utility of the genetic approach.
Arterial hypotension triggers perifocal depolarizations and aggravates secondary damage in focal brain injury
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Traumatic brain lesions, however, may differ in many aspects from focally ischemic lesions. They trigger perifocal depolarizations (PFDs) in a much lower frequency than seen after experimental stroke (Back et al., 1994; Hermann et al., 1998; Hermann et al., 1999b; Nedergaard and Astrup, 1986), and the surroundings of focal traumatic injury do not show a major disturbance of protein synthesis or pH as seen in peri-infarct regions (Back et al., 2000; Hermann et al., 1999a; Mies et al., 1991). In addition, secondary insults like hypotension, hypoxia and intracranial hypertension occur more frequently in neurotrauma patients than after stroke (Chesnut et al., 1993).
Perifocal depolarizations (PFD) have been observed after traumatic brain injury, are known to disturb cerebrovascular reactivity and thus may contribute to the morphological consequences of brain injury. In this investigation, the role of PFD was studied in focal brain lesions with/without induction of delayed hypotension. Cerebral freeze lesions were induced in anesthetized normotensive rats that underwent perfusion fixation of brains 5 min, 4 h or 24 h after lesioning, respectively, to obtain quantitative histopathology. In additional groups, a 45-min period of moderate hypobaric hypotension was applied 15 min post-trauma and brains were perfusion fixed after 4 h or 24 h. In a second series, the direct current (DC) potential and cortical laser-Doppler flow (LDF) were measured adjacent to lesions under normotensive or hypotensive conditions. Sham procedures were carried out in rats that underwent hypotension alone. Lesioning resulted in a significant LDF decrease to 50% of baseline, further decreased during hypotension to less than 40% of control (P < 0.05). Sham animals had LDF values between 60 and 70% of control when subjected to hypotension. Focal brain injury always induced a negative DC shift shortly after lesioning. In 6 of 8 rats that underwent cold lesion plus hypotension, a second PFD was observed approximately 2.5 min after onset of hypotension accompanied by a relative LDF increase by 25 ± 12%. Lesion expansion was significantly worsened by hypotension (8.19 ± 0.56 mm³ at 24 h) compared with normotensive rats (7.01 ± 0.3 mm³ at 24 h, P < 0.01). We conclude that hypotension triggers depolarizations by an ischemic mechanism that contributes to final tissue damage.
Expression of c-jun, mitogen-activated protein kinase phosphatase-1, caspase-3 and glial fibrillary acidic protein following cortical cold injury in rats: Relationship to metabolic disturbances and delayed cell death
2004, Neuroscience
The expression of c-jun, mitogen-activated protein kinase phosphatase-1 (mkp-1), caspase-3 and glial fibrillary acidic protein (gfap) was examined at 1, 3 and 7 days after cortical cold injury in rats by in situ hybridisation and immunocytochemistry. Alterations of gene expression were related to metabolic disturbances and delayed cell death, as revealed by cerebral protein synthesis autoradiography, ATP bioluminescence, pH fluorescence and terminal transferase biotinylated dUTP nick end labelling (TUNEL). Protein synthesis autoradiographies depicted sharply demarcated cortex lesions, which were almost congruent with areas exhibiting ATP depletion (lesion volume: 16.9±11.8 mm³ after 7 days). Lesions were surrounded by a region of tissue alkalosis, which was most prominent 1 day after trauma. Delayed cell injury, as revealed by TUNEL, was noticed in a thin rim around the lesion border on day 1 (tissue volume: 1.7±0.8 mm³) and, to lesser extent, days 3 and 7 post-lesioning. However, only a small percentage of cells in this area were positive for activated caspase-3 protein. TUNEL(+) cells were further seen in the ventrobasal thalamus after 7 days. In the thalamus, the appearance of DNA-fragmented cells was closely accompanied by activated caspase-3 expression. In situ hybridisations revealed that cell injury both in the peri-lesion rim and ventrobasal thalamus was associated with increased c-jun and gfap, but not mkp-1 and caspase-3 mRNA levels. Gene responses were not confined to areas revealing irreversible cell death: mkp-1 mRNA was bilaterally upregulated in the lesion-remote entorhinal cortex, cingulate cortex and reticular thalamus at 7 days after trauma, and caspase-3 mRNA was slightly, but significantly downregulated in the entorhinal cortex after 3 and 7 days. Gfap mRNA was elevated in all regions exhibiting tissue alkalosis. Our data suggest that delayed cell injury after cortex trauma may be apoptotic in the ventrobasal thalamus, but not the peri-lesion rim. The dissociated responses of c-jun, mkp-1 and caspase-3 mRNAs may represent important factors influencing tissue viability.
Biphasic expression of activating transcription factor-3 in neurons after cerebral infarction
2003, Molecular Brain Research
It has been demonstrated that some of immediate early genes such as c-Jun are induced immediately and transiently following focal cerebral ischemia. Here we newly characterize the activating transcription factor (ATF)-3 as a focal ischemia associated immediate early gene. Using in situ hybridization and immunohistochemistry, we compared the expression profile of ATF-3 with those of ATF-2 and c-Jun after middle cerebral artery (MCA) occlusion. Focal cerebral ischemia induced two temporal and spatial patterns of ATF-3 expression. Early and transient induction of ATF-3 mRNA was observed in the core and margins of the cortex immediately after MCA occlusion. Late-onset and prolonged expression of ATF-3 mRNA and its protein were specifically identified in the peri-infarct cortex and thalamus where neurons survive at least 1 month. The expression profiles of ATF-3 and c-Jun were virtually similar, but c-Jun expression was also observed in other regions of the brain in control rats. Expression of ATF-2 was ubiquitously seen in neuronal cells throughout the brain in normal rats, but was suppressed in ischemic regions. Double immunohistochemical labeling revealed concurrent expression of ATF-3 and phospho-c-Jun in neurons. We conclude that the transcription factor ATF-3 is a suitable marker of neurons subjected to ischemic insult directly and indirectly, and that cooperative works of ATF-3 and c-Jun may be crucial triggers of various transcriptional responses to the ischemic insult.
Cortical spreading depression transiently activates MAP kinases
2002, Molecular Brain Research
Cortical spreading depression (CSD) has been shown to have neuroprotective effects when administered in advance of cerebral ischemia. The mechanism by which CSD induces its neuroprotective effect however remains to be elucidated. Since MAP kinases have been shown to impart neuroprotection in ischemic preconditioning paradigms, we attempted to determine the role CSD may have in the activation of MAPK. We show that CSD is capable of increasing the phosphorylation of ERK in a MEK-dependent manner. This phosphorylation is, however, transient, as phosphorylated ERK levels return to control levels 45 min after 2 h of CSD elicitation. Immunohistochemical analysis reveals that the phosphorylated form of ERK is located ubiquitously in cells of the CSD-treated cortex while CSD-elicited MEK phosphorylation resides solely in the nuclei. These data suggest that CSD may act via the MAP kinase pathways to mediate preconditioning.
Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice
2001, Neuroscience
The evolution of brain injury was examined in mice subjected to focal cerebral ischemia as induced by 30 min of intraluminar thread occlusion of the middle cerebral artery, followed by 3 h to 3 days of reperfusion. Metabolic dysfunctions were studied by ³H-leucine autoradiography for the measurement of cerebral protein synthesis and by regional ATP bioluminescent imaging. Metabolic changes were compared with responses of the genes c-fos, c-jun, heat-shock protein gene (hsp)72, p53-activated gene (pag)608 and caspase-3, which were investigated by in situ hybridization histochemistry and immunocytochemistry, and correlated with the degree of DNA fragmentation, as assessed by the terminal TdT-mediated dUTP-biotin nick end labeling method. Intraluminar thread occlusion led to a reproducible reduction of cerebral laser Doppler flow to 20–30% of control. Thread withdrawal was followed by a short-lasting post-ischemic hyperperfusion to approximately 120%. In non-ischemic control animals, fractional protein synthesis values of 0.81±0.26 and 0.94±0.23 were obtained. Thread occlusion resulted in a suppression of protein synthesis throughout the territory of the middle cerebral artery after 3 h of reperfusion (0.04±0.08 in caudate–putamen and 0.14±0.19 in somatosensory cortex, P<0.05). Protein synthesis partly recovered in the cortex after 24 h and 3 days (0.71±0.40 and 0.63±0.26, respectively), but remained suppressed in the caudate–putamen (0.14±0.22 and 0.28±0.28). Regional ATP levels did not show any major disturbances at the reperfusion times examined. Thread occlusion resulted in a transient increase of c-fos mRNA levels in ischemic and non-ischemic parts of the cortex and caudate–putamen at 3 h after ischemia, which suggests that spreading depressions were elicited in the tissue. At the same time, c-jun and hsp72 mRNAs were elevated only in ischemic brain areas showing inhibition of protein synthesis. C-fos and c-jun responses completely disappeared within 24 h of reperfusion. Hsp72 mRNA levels remained elevated in the cortex after 24 h, but decreased to basal values in the caudate–putamen. Twenty-four hours after reperfusion, pag608 and caspase-3 mRNA levels increased in the caudate–putamen, where protein synthesis rates were still reduced, and remained elevated even after 3 days. However, pag608 and caspase-3 mRNA levels did not increase in the cortex, where protein synthesis recovered. After 24 h and 3 days, functionally active p20 fragment of caspase-3 was detected in the caudate–putamen, closely associated with the appearance of DNA fragmented cells. Neither activated caspase-3 nor DNA fragmentation were noticed in the cortex.
In summary, the suppression of protein synthesis is reversible in the ischemia-resistant cortex following 30 min of thread occlusion in mice, but persists in the vulnerable caudate–putamen. In the caudate–putamen, apoptotic programs are induced, closely in parallel with the manifestation of delayed cell death. Thus, the recovery of protein synthesis may be a major factor influencing tissue survival after transient focal ischemia.

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Expression of c-fos, junB, c-jun, MKP-1 AND hsp72 following traumatic neocortical lesions in rats—relation to spreading depression

Abstract

Section snippets

Animal experimentation

Physiological variables

Laser Doppler flowmetry and electrophysiological observations

Discussion

Conclusions

Acknowledgements

Neuron

Expl Neurol.

Brain Res. Rev.

Molec. Brain Res.

Neurosci. Lett.

Trends Neurosci.

Brain Res.

Brain Res.

Brain Res.

Molec. Brain Res.

Molec. Brain Res.

Expl Neurol.

Cell

Brain Res.

Brain Res.

Oncogene jun encodes a sequence-specific trans-activator similar to AP-1

Nature

Signal transduction via the MAP kinases: proceed at your own RSK

Proc. natn. Acad. Sci. U.S.A.

cDNA sequence of a growth factor-inducible immediate early gene and characterization of its encoded protein

Oncogene

Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase

Proc. natn. Acad. Sci. U.S.A.

Isolation and characterization of the c-fos (rat) cDNA and analysis of post-translational modification in vitro

Oncogene

Photothrombotic infarction triggers multiple episodes of cortical spreading depression in distant brain regions

J. cerebr. Blood Flow Metab.

Protein synthesis inhibitors differentially superinduce c-fos and c-jun by three distinct mechanisms: lack of evidence for labile repressors

Eur. molec. Biol. Org. J.

Spreading EEG depression in the neo-, paleo- and archicortical structures of the brain of the rat

Physiol bohemoslov

Regulation of c-fos gene expression in hamster fibroblasts: initiation and elongation of transcription and mRNA degradation

Nucl. Acids Res.