Research reportBenign focal ischemic preconditioning induces neuronal Hsp70 and prolonged astrogliosis with expression of Hsp27
Introduction
Ischemic injury induces elevated expression of heat shock proteins in the brain [18], [21], [60], [63], [68]. Heat shock proteins contribute to the cellular repair processes by re-folding denatured proteins [49] and acting as molecular chaperones in normal processes such as protein translocation and folding [8], [22], [59]. The highly inducible member of the 70-kDa family of Hsps, Hsp70, has been associated with resistance (i.e. tolerance) to ischemic injury in the heart [14], [15], [41], [51], [55], [56] and brain [20], [29], [30], [31], [36], [37], [42], [48], [55], [64], [73].
Brain tolerance to ischemic injury (i.e. ischemic tolerance; IT) has been observed following heat shock treatment [30] and mild ischemic preconditioning (PC) treatments. A mild global ischemic insult can reduce the extent of hippocampal damage after a subsequent severe global ischemic insult [31], [36] and significantly decrease the infarct size after permanent middle cerebral artery occlusion [64]. Similarly, a brief focal ischemic episode has been shown to be protective against subsequent severe global ischemic injury [20]. In addition, brief repetitive middle cerebral artery occlusion can decrease infarct size following a subsequent 100-min occlusion, however Hsp70 accumulation and degradation did not match the acquisition and decay of ischemic tolerance [12].
Other heat shock proteins also have been implicated in cellular resistance to injury. The 27-kDa heat shock protein (Hsp27) has been shown to increase cell resistance to oxidative injury [44] or thermal stress [34]. Recently, Hsp27 has been shown to be highly inducible in the neocortex of rats after seizure activity [52], photothrombotic injury [53] and cortical spreading depression [23], [25], [54]. In fact, Hsp27 also has been shown to be expressed following cerebral ischemia [27], [28].
We have established a focal PC paradigm that produces significant and prolonged IT of the brain to subsequent permanent MCAO. In this model, an initial 10 min of MCAO (i.e. PC) induces a brain tolerance that persists from 1 to 7 days of reperfusion [6]. Under these conditions, both infarct size and neurological deficits are significantly (greater than 50%) reduced following permanent MCA occlusion. These robust protective effects are dependent on de novo protein synthesis that occurs after PC but not on newly synthesized proteins after IT is established [6]. The purpose of the present study was to characterize the temporal and cellular distribution of Hsp70 and Hsp27 expression and astrocytic changes that occur following PC, and their relationships to the induced brain tolerance using this model. Northern analyses of message expression and immunohistologic staining for protein expression post-PC were determined. Double cellular immunohistologic labeling using glial fibrillary acidic protein (GFAP) was also performed to confirm glial activation induced by PC and its association with IT and the increased expression of Hsp27. The data demonstrate that the very brief and benign ischemia used for PC that induces significant neuroprotection of the brain is also associated with a very significant and prolonged astrogliosis and a robust increased expression of Hsp27 in these activated astrocytes. Expression of Hsp70 is less prolonged and is mainly confined to neuronal cells following PC. Although no brain injury is produced by PC, clearly many cellular and protein synthetic changes are produced associated with brain tolerance.
Section snippets
Animals
Adult male spontaneously hypertensive rats (290–350 g; n=43) were utilized in the present studies. Prior to surgical procedures, the rats were anesthetized with sodium pentobarbital (60 mg/kg intraperitoneally). All the animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Procedures and the protocol for using laboratory animals in the present studies were
Expression of Hsp70 and Hsp27 mRNA after PC
A representative autoradiograph of Northern blot for HSP-70 and HSP-27 mRNA expression in the cortex at selected time points after PC and in sham-operated cortical samples is illustrated in Fig. 1. The quantitative data for HSP-70 and HSP-27 mRNA (n=6), after normalizing to a house-keeping gene rpL32, are summarized graphically in Fig. 2. Sham-operated samples were taken from 6 (n=3) and 24 h (n=3) post-surgery. Expression at both timepoints were similar and the data was pooled. Practically no
Discussion
In this study, PC transiently upregulated Hsp70 mRNA, but produced a later more prolonged upregulation of Hsp27. Hsp27 mRNA expression was particularly sensitive to brain stimulation (i.e. non-significant trend to increase just due to surgery alone). These data were consistent with protein expression data. An early elevated neuronal Hsp70 immunoreactivity was observed in the ischemic area and a later, remarkable Hsp27 astrologic immunoreactivity within and outside the ischemic area was observed
Acknowledgements
The authors would like to thank Sue Tirri for secretarial assistance. RWC was the recipient of a Visiting Scientist Award from Heart and Stroke Foundation of Canada and was spending his 1996–97 sabbatical leave at SmithKline Beecham from Dalhousie University during this research. At Dalhousie University, RWC’s research is funded by the Heart and Stroke Foundation of New Brunswick. GZF and XKW are now located at DuPont Pharmaceuticals Co., Cardiovascular Research, Wilmington, DE. These data were
References (74)
- et al.
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
Neuroscience
(1996) - et al.
Proteomics: Quantitative and physical mapping of cellular proteins
Trends Biotechnol.
(1999) - et al.
Induction of cortical spreading depression with potassium chloride upregulates levels of messenger RNA for glial fibrillary acidic protein in cortex and hippocampus: inhibition by MK-801
Brain Res.
(1993) - et al.
The role of extracellular ionic changes in upregulating the mRNA for glial fibrillary acidic protein following spreading depression
Brain Res.
(1995) - et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987) - et al.
The role of apoptosis in sexual differentiation of the rat sexually dimorphic nucleus of the preoptic area
Brain Res.
(1996) - et al.
Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia
Brain Res. Bull.
(1991) - et al.
Application of potassium chloride to the brain surface induces the c-fos proto-oncogene: reversal by MK-801
Brain Res.
(1990) - et al.
MK-801 affects the potassium-induced increase of glial fibrillary acidic protein immunoreactivity in rat brain
Brain Res.
(1992) - et al.
Astroglial and microglial reactions in the gerbil hippocampus with induced ischemic tolerance
Brain Res.
(1994)
Immunohistochemical localization of the low molecular weight stress protein Hsp27 following focal cerebral ischemia in the rat
Brain Res.
Induction of 27-kDa heat shock protein following cerebral ischemia in a rat model of ischemic tolerance
Brain Res.
‘Ischemic tolerance’ phenomenon detected in various brain regions
Brain Res.
Protection of rat hippocampus against ischemic neuronal damage by pretreatment with sublethal ischemia
Brain Res.
Temporal profile of heat shock protein 70 synthesis in ischemic tolerance induced by preconditioning ischemia in rat hippocampus
Neuroscience
Astrocytic demise precedes neuronal death in focal ischemic rat brain
Mol. Brain Res.
The release and uptake of excitatory amino acids
Trends Pharmacol. Sci.
Ischemic tolerance due to the induction of HSP70 in a rat ischemic recirculation model
Brain Res.
Speculations on the functions of the major heat shock and glucose regulated proteins
Cell
Expression of the 27-kDa heat shock protein (Hsp27) following kainic acid-induced status epilepticus in the rat
Neuroscience
Differential expression of c-fos, hsp70 and hsp27 after photothrombotic injury in the rat brain
Mol. Brain Res.
Glutathione is present in high concentrations in cultured astrocytes but not in cultured neurons
Brain Res.
Interleukin-1 receptor antagonist inhibits ischemic and excitotoxic damage in the rat
Brain Res. Bull.
Heat shock protein protection
Trends Neurosci.
Prior ischemic stress protects against experimental stroke
Neurosci. Lett.
A comparison of induced heat-shock protein in neurons destined to survive and those destined to die after transient ischemia in rats
Brain Res.
Astrocytes protect cultured neurons from degeneration induced by anoxia
Brain Res.
Role of glial cells in the regulation of the brain ion microenvironment
Prog. Neurobiol.
Gene expression and induced ischemic tolerance following brief insults
Acta Neurobiol. Exp. Warsz.
Inflammatory mediators and stroke: New opportunities for novel therapeutics
J. Cereb. Blood Flow Metab.
Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: myeloperoxidase activity assay and histologic verification
J. Neurosci. Res.
Genetic hypertension and increased susceptibility to cerebral ischemia
Neurosci. Biobehav. Rev.
Ischemic preconditioning and brain tolerance: Temporal, histologic and functional outcomes, protein synthesis requirement and IL-1ra and early gene expression
Stroke
Neuron specific enolase increases in cerebral and systemic circulation following focal ischemia
Brain Res.
Heat-shock proteins as molecular chaperones
Eur. J. Biochem.
Stress proteins and tolerance to focal cerebral ischemia
J. Cereb. Blood Flow Metab.
Trauma-induced protein in rat tissues: A physiological role for a ‘heat shock’ protein?
Science
Cited by (149)
The effect of female sex hormones on Hsp27 phosphorylation and histological changes in prefrontal cortex after tMCAO
2021, Pathology Research and PracticeNeuroprotective effects of a novel carnosine-hydrazide derivative on hippocampal CA1 damage after transient cerebral ischemia
2019, European Journal of Medicinal Chemistry