Differential antioxidant response between two Symbiodinium species from contrasting environments.

High sea surface temperature accompanied by high levels of solar irradiance is responsible for the disruption of the symbiosis between cnidarians and their symbiotic dinoflagellates from the genus Symbiodinium. This phenomenon, known as coral bleaching, is one of the major threats affecting coral reefs around the world. Because an important molecular trigger to bleaching appears related to the production of reactive oxygen species (ROS), it is critical to understand the function of the antioxidant network of Symbiodinium species. In this study we investigated the response of two Symbiodinium species, from contrasting environments, to a chemically induced oxidative stress. ROS produced during this oxidative burst reduced photosynthesis by 30 to 50% and significantly decreased the activity of superoxide dismutase. Lipid peroxidation levels and carotenoid concentrations, especially diatoxanthin, confirm that these molecules act as antioxidants and contribute to the stabilization of membrane lipids. The comparative analysis between the two Symbiodinium species allowed us to highlight that Symbiodinium sp. clade A temperate was more tolerant to oxidative stress than the tropical S. kawagutii clade F. These differences are very likely a consequence of adaptation to their natural environment, with the temperate species experiencing conditions of temperature and irradiance much more variable and extreme.

Focal cerebral ischemia impairs motivation in a progressive FR schedule of reinforcement in mice.

Characterization of functional outcome in animal stroke models is essential to improve preclinical drug screenings. Operant procedures showed promising results for the identification of long-lasting functional deficits. In particular, a suppression of lever-pressing in high ratio schedules has been consistently found in rodent models of ischemic stroke. In the present work, we attempted to replicate these isolated observations, by submitting C57Bl/6J mice to a progressive fixed-ratio schedule of reinforcement three weeks after MCAO or sham surgery. Results showed a significant lever-pressing impairment in the MCAO group. Motivational factors (longer post-reinforcement pause, lesser appeal for food rewards) seemed accountable for the deficit, while motor abilities appeared preserved. These findings resemble fatigue-like states experienced by stroke survivors and may be used as long-term measures of behavioral outcome following experimental stroke.

Hyperthermic induction of the 27-kDa heat shock protein (Hsp27) in neuroglia and neurons of the rat central nervous system.

The 27-kDa heat shock protein (Hsp27) is constitutively expressed in many neurons of the brainstem and spinal cord, is strongly induced in glial cells in response to ischemia, seizures, or spreading depression, and is selectively induced in neurons after axotomy. Here, the expression of Hsp27 was examined in brains of adult rats from 1.5 hours to 6 days after brief hyperthermic stress (core body temperature of 42 degrees C for 15 minutes). Twenty-four hours following hyperthermia, Western blot analysis showed that Hsp27 was elevated in the cerebral cortex, hippocampus, cerebellum, and brainstem. Immunohistochemistry for Hsp27 revealed a time-dependent, but transient, increase in the level of Hsp27 immunoreactivity (Hsp27 IR) in neuroglia and neurons. Hsp27 IR was detected in astrocytes throughout the brain and in Bergmann glia of the cerebellum from 3 hours to 6 days following heat shock. Peak levels were apparent at 24 hours, gradually declining thereafter. In addition, increases in Hsp27 IR were detected in the ependyma and choroid plexus. Hyperthermia induced Hsp27 IR in neurons of the subfornical organ and the area postrema within 3 hours and reached a maximum by 24 hours with a return to control levels 4-6 days after hyperthermia. Specific populations of hypothalamic neurons also showed Hsp27 IR after hyperthermia. These results demonstrate that hyperthermia induces transient expression of Hsp27 in several types of neuroglia and specific populations of neurons. The pattern of induced Hsp27 IR suggests that some of the activated cells are involved in physiological responses related to body fluid homeostasis and temperature regulation.

Induction of the 27-kDa heat shock protein (Hsp27) in the rat medulla oblongata after vagus nerve injury.

The 27-kDa heat shock protein (Hsp27) is constitutively expressed in motor and sensory neurons of the brainstem. Hsp27 is also rapidly induced in the nervous system following oxidative and cellular metabolic stress. In this study, we examined the distribution of Hsp27 in the rat medulla oblongata by means of immunohistochemistry after the vagus nerve was cut or crushed. After vagal injury, rats were allowed to survive for 6, 12, 24 h, 2, 4, 7, 10, 14, 30, or 90 days. Vagus nerve lesions resulted in a time-dependent up-regulation of Hsp27 in vagal motor and nodose ganglion sensory neurons that expressed Hsp27 constitutively and de novo induction in neurons that did not express Hsp27 constitutively. In the dorsal motor nucleus of the vagus nerve (DMV) and nucleus ambiguus, the levels of Hsp27 in motor neurons were elevated within 24 h of injury and persisted for up to 90 days. Vagal afferents to the nucleus of the tractus solitarius (NTS) and area postrema showed increases in Hsp27 levels within 4 days that were still present 90 days postinjury. In addition, increases in Hsp27 staining of axons in the NTS and DMV suggest that vagus nerve injury resulted in sprouting of afferent axons and spread into areas of the dorsal vagal complex not normally innervated by the vagus. Our observations are consistent with the possibility that Hsp27 plays a role in long-term survival of distinct subpopulations of injured vagal motor and sensory neurons.

Transgenic mice expressing the human inducible Hsp70 have hippocampal neurons resistant to ischemic injury.

Using transgenic mice constitutively expressing the human inducible Hsp70, we examined the role of Hsp70 on cell survival after focal cerebral ischemia. Twenty-four hours after permanent occlusion of the middle cerebral artery, no difference in infarct area was detected between Hsp70-transgenic and non-transgenic mice. In the non-transgenic mice, many pyramidal neurons of the ipsilateral hippocampus were observed to be pyknotic. However, in all Hsp70-transgenic mice, hippocampal pyramidal neurons showed normal morphology and no evidence of pyknosis. This suggests that constitutive expression of Hsp70 reduces the extent of damage following permanent middle cerebral artery occlusion.

Constitutive expression of the 27-kDa heat shock protein (Hsp27) in sensory and motor neurons of the rat nervous system.

In this study, the constitutive expression of the 27-kDa heat shock protein (Hsp27) in the adult rat central nervous system has been examined by immunohistochemistry and by two-dimensional gel Western blot analysis. Hsp27 immunoreactivity was observed primarily in motoneurons of cranial nerve nuclei and spinal cord, and in primary sensory neurons and their central processes. Also, Hsp27 immunoreactivity was present in neurons of the arcuate nucleus and of the reticular formation. However, only a subset of these neurons was Hsp27-immunoreactive. Most general somatic efferent motoneurons of the hypoglossal nucleus and spinal motor columns and most special visceral efferent motoneurons of the cranial nerve nuclei were Hsp27-positive. In contrast, fewer general somatic efferent motoneurons for eye muscles were Hsp27-positive, and only a small proportion of general visceral efferent neurons, i.e., parasympathetic and sympathetic preganglionic neurons, were stained for Hsp27. Many pseudounipolar sensory neurons were Hsp27-immunoreactive, and the patterns of staining in central sensory nuclei suggested that specific subpopulations of sensory neurons contained Hsp27. The cellular distribution of Hsp27 was uniform throughout the cytoplasm, including the perikaryon, axon and dendrites, the latter often exhibiting varicosities or beading in distal processes. Western blot analyses revealed that at least three phosphorylated isoforms of Hsp27 were present in the spinal cord. These results suggest that constitutively expressed Hsp27 may be related to functional subpopulations of motoneurons and primary sensory neurons.

Cortical application of potassium chloride induces the low-molecular weight heat shock protein (Hsp27) in astrocytes.

Spreading depression induces tolerance to ischemic injury, and ischemic tolerance has been associated with expression of heat shock proteins (Hsp). Here we examine Hsp27 expression after KCl-induced spreading depression. Twenty-minute cortical KCl application induced Hsp27 immunoreactivity in glial fibrillary acidic protein-positive astrocytes of the ipsilateral neocortex. Systemic administration of MK-801 (3 mg/kg) suppressed KCl-induced Hsp27 expression in the parietal cortex. Astrocytes in the posterior cingulate and retrosplenial cortex did not express Hsp27 after KCl application but did express Hsp27 after systemic administration of high dose MK-801 (9 mg/kg). Whereas Hsp27 was usually observed in all layers of the parietal cortex after 5-minute application of KCl, in 2 of 6 rats, Hsp27 was seen in clusters of astrocytes or in astrocytes in the superficial layers I to III of the parietal cortex. We conclude that (1) cortical application of KCl triggered Hsp27 astrocytic expression; (2) astrocytes in the cingulate and retrosplenial cortex responded differently compared with astrocytes of the parietal cortex; (3) Hsp27 expression progressed from small clusters of astrocytes throughout superficial layers of the cortex that joined and recruited astrocytes in deeper layers; (4) several mechanisms induced Hsp27 astrocytic expression. We propose that Hsp27 is involved in spreading depression-induced ischemic tolerance through protection of astrocyte function.

Differential expression of c-fos, Hsp70 and Hsp27 after photothrombotic injury in the rat brain.

In situ hybridization and immunohistochemistry were used to examine the expression of c-fos, Hsp70 and Hsp27 following photothrombotic injury in the right fronto-parietal cortex of the rat. C-fos mRNA and protein were detected in the entire cerebral cortex on the lesioned side. Hsp70 mRNA accumulation was observed only adjacent and peripheral to the site of the lesion. At 1 h after photothrombotic injury, Hsp70 expression delineates the area of necrosis at 24 h after photothrombotic injury. Hsp27 protein was observed in the ipsilateral cerebral cortex with the exception of the deep layers of the cingulate cortex. In addition, while c-Fos immunoreactivity was localized in cell nuclei, Hsp27 immunoreactivity was detected in the cytoplasm of astrocytes. These results demonstrate that unilateral cortical injury induces changes in gene expression that vary according to cell type and brain region.

A novel seizure-induced synaptotagmin gene identified by differential display.

Systemic administration of kainic acid, a cyclic analogue of glutamate, produces many of the clinical features of human temporal lobe epilepsy and status epilepticus in rats, including the induction of motor convulsions and the degeneration of neurons in the hippocampus and piriform cortex. Differential display PCR was used to identify mRNAs that are differentially expressed between degenerating and nondegenerating tissues in the brain after kainic acid-induced seizure activity. A novel cDNA fragment expressed in the degenerating hippocampus and piriform cortex, but not in the nondegenerating parietal cortex, was identified, cloned, and sequenced. This novel cDNA fragment identified a new member of the synaptotagmin gene family that is rapidly and transiently induced in response to seizure activity. Differential expression of this synaptotagmin gene, syt X, was confirmed by Northern blot analysis and in situ hybridization. This novel, inducible synaptotagmin gene may provide a direct link between seizure-induced neuronal gene expression and subsequent modulation of synaptic structure and function.

Expression of the 27,000 mol. wt heat shock protein following kainic acid-induced status epilepticus in the rat.

Western analysis and immunohistochemistry were used to determine the time-course and the distribution of the 27,000 mol. wt heat shock protein, Hsp27, in rat brain following systemic administration of kainic acid. No Hsp27 immunoreactivity was detected in naive control animals or in rats that failed to develop status epilepticus. Hsp27 immunoreactivity was detected as early as 12 h in the parietal cortex, piriform cortex and the hippocampus of rats that developed status epilepticus. The number of cells expressing Hsp27 and the intensity of Hsp27 immunoreactivity were increased 24 h after kainic acid administration. Hsp27 immunoreactivity was still observed seven days post-kainic acid injection. The morphology of the Hsp27-positive cells and double immunofluorescence against Hsp27 and glial fibrillary acidic protein revealed that Hsp27-positive cells were astrocytes. In addition, the distribution of Hsp27 suggested that astrocytic Hsp27 was dependent on excitation-induced metabolic stress rather than the direct effect of kainic acid on astrocytes.

The inducible 70,000 molecular/weight heat shock protein is expressed in the degenerating dentate hilus and piriform cortex after systemic administration of kainic acid in the rat.

Using both immunohistochemistry and in situ hybridization, we examined the rat brain for the expression of the inducible 70,000 mol. wt heat shock protein, Hsp70, at 3,6,12 and 24 h after systemic administration of kainic acid. In contrast to previous reports, the present study demonstrates that neurons in the regions most susceptible to seizure-induced cell death accumulate both Hsp70 messenger RNA and protein. Neurons in the denate hilus and piriform cortex contained Hsp70 messenger RNA at 6 h and protein at 12 h. These neutrons contained little or no Hsp70 messenger RNA or protein at 24 h when the majority of cells in these area were pyknotic. Injured neurons in areas such as the parietal cortex, which are less susceptible to seizure-induced cell death, expressed and maintained high levels of Hsp70 messenger RNA and protein at 12 and 24 h. This work suggest that Hsp70 messenger RNA and protein are rapidly and transiently expressed in dying neurons, and contradicts the notion that Hsp70 only accumulates in injured neurons that survive.

Differential accumulation of mRNA for immediate early genes and heat shock genes in heart after ischaemic injury.

Ischaemic injury leads to the expression of heat shock and immediate early genes. Here the localization of this induction is examined by in situ hybridization analysis in serial sections of buffer-perfused isolated rat heart after 30 min of coronary artery occlusion. The accumulation of mRNA for hsc70, hsp70, c-fos, c-jun, and Erg-1 was localized coincidently and was restricted to the ischaemic area of the heart. mRNA for these genes was undetectable at the end of the ischaemic period (no reperfusion). After 30 min of reperfusion, accumulation of mRNA for hsc70, hsp70, c-fos, and c-jun was detectable and increased with further reperfusion. Within the area labelled for these gene products was a central area of less intense labelling which corresponded to the necrotic zone. The immediate early gene product, jun-B, was localized in both the ischaemic and the non-ischaemic area of the hearts. These results suggest that the area of the heart where heat shock and immediate early gene transcripts accumulate, while injured, recovers transcriptional activity, and that the central area where minimal heat shock and immediate early gene transcripts accumulate, does not recover transcriptional activity and is irreversibly injured.

Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery.

Heat shock treatment induces expression of several heat shock proteins and subsequent post-ischemic myocardial protection. Correlations exist between the degree of stress used to induce the heat shock proteins, the amount of the inducible heat shock protein 70 (HSP70) and the level of myocardial protection. The inducible HSP70 has also been shown to be protective in transfected myogenic cells. Here we examined the role of human inducible HSP70 in transgenic mouse hearts. Overexpression of the human HSP70 does not appear to affect normal protein synthesis or the stress response in transgenic mice compared with nontransgenic mice. After 30 min of ischemia, upon reperfusion, transgenic hearts versus nontransgenic hearts showed significantly improved recovery of contractile force (0.35 +/- 0.08 versus 0.16 +/- 0.05 g, respectively, P < 0.05), rate of contraction, and rate of relaxation. Creatine kinase, an indicator of cellular injury, was released at a high level (67.7 +/- 23.0 U/ml) upon reperfusion from nontransgenic hearts, but not transgenic hearts (1.6 +/- 0.8 U/ml). We conclude that high level constitutive expression of the human inducible HSP70 plays a direct role in the protection of the myocardium from ischemia and reperfusion injury.