Elsevier

Brain Research

Volume 863, Issues 1–2, 28 April 2000, Pages 169-181
Brain Research

Research report
Benign focal ischemic preconditioning induces neuronal Hsp70 and prolonged astrogliosis with expression of Hsp27

https://doi.org/10.1016/S0006-8993(00)02133-8Get rights and content

Abstract

We have established a focal preconditioning (PC) paradigm that produces significant and prolonged ischemic tolerance (IT) of the brain to subsequent permanent middle cerebral artery occlusion (MCAO). PC using 10 min of MCAO induces brain tolerance at 1–7 days of reperfusion that requires active protein synthesis. The protective protein(s) involved are unknown. In these studies the increased transcription and translation of the inducible 70-kDa heat shock protein (Hsp70) and the 27-kDa heat shock protein (Hsp27), and astrogliosis/glial fibrillary acidic protein (GFAP) were determined by Northern analysis and immunohistochemistry following PC. Cellular localization of proteins was determined by double labeling. PC produced no brain injury but did increase Hsp70 mRNA transiently at 6 h and increased Hsp27 mRNA later at 24 h for at least 5 days. Protein expression induced by PC exhibited a similar profile. Hsp70 protein was primarily expressed in neurons from 1 to 5 days post-PC throughout the PC cortex. Hsp27 protein expression was initiated later for a much longer period of time. A remarkable astroglyosis was verified with increased astrocytic Hsp27 from 1 to 7 days after PC. Gliosis with increased Hsp27 in the PC cortex was still present but reduced 4 weeks after PC. Therefore, PC that results in brain tolerance/neuroprotection increases neuronal Hsp70 in the PC cortex and activated astrocytic Hsp27 in the PC cortex in a temporal fashion associated with developing IT. The short duration of benign ischemia (PC) that produces IT produces a robust, long-lived cellular and protein synthetic response that extends throughout the entire cortex (i.e. well beyond the MCA perfusion territory). The resulting IT is associated with changes in astrocyte-activation that might provide increased support and protection from injury. Although both Hsp70 and Hsp27 may participate in the neuroprotection/brain tolerance induced by PC, the temporal expression patterns of these proteins indicate that they are not solely responsible for the tolerance to brain injury.

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

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