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Stroke. 1996;27:504-513

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(Stroke. 1996;27:504-513.)
© 1996 American Heart Association, Inc.

Articles

Subarachnoid Injections of Lysed Blood Induce the hsp70 Stress Gene and Produce DNA Fragmentation in Focal Areas of the Rat Brain

Paul Matz, MD; Philip Weinstein, MD; Bradley States, BS; Jari Honkaniemi, MD, PhD Frank R. Sharp, MD

From the Departments of Neurosurgery (P.M., P.W., B.S.) and Neurology (J.H., F.R.S.), University of California at San Francisco, and Veterans Affairs Medical Center, San Francisco, Calif; and Department of Neurology (J.H.), University of Tampere (Finland).

Correspondence to Dr Paul Matz, Box 0112, Department of Neurosurgery M787, 786 Moffitt Hospital, University of California at San Francisco, San Francisco, CA 94143-0112. E-mail matzp@neuro.ucsf.edu.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
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Background and Purpose Most experimental studies of subarachnoid hemorrhage have demonstrated little histological evidence of injury. In the present study we examined both the expression of the hsp70 heat-shock gene, a molecular marker of reversible neuronal injury, and DNA fragmentation, a marker of irreversible cell injury and death.

Methods Lysed blood, whole blood, oxyhemoglobin, bovine serum albumin, and saline were injected into the cisterna magna of adult rats. The induction of hsp70 mRNA and HSP70 heat-shock protein was assessed with the use of in situ hybridization and immunocytochemistry, respectively. Fragmentation of genomic DNA was studied by DNA nick end-labeling with the use of terminal deoxynucleotidyl transferase and biotinylated dATP.

Results Expression of the hsp70 gene was not induced in the brains of rats injected with whole blood, oxyhemoglobin, bovine serum albumin, or saline. Lysed blood injections, however, induced hsp70 mRNA at 6 and 24 hours in the cerebellar hemispheres and in focal regions of the basal forebrain. HSP70 protein was induced by 24 hours and persisted for at least 4 days in the same regions. HSP70 protein was localized to patches of glial cells and occasional neurons in the forebrain. In the cerebellum HSP70 was localized to Bergmann glial cells, granule cells, molecular layer stellate cells, and occasional Purkinje cells. DNA nick end-labeling showed patches of labeled cells in the basal forebrain that occurred in the same regions that hsp70 mRNA was induced.

Conclusions The results demonstrate focal stress gene induction and DNA fragmentation after subarachnoid hemorrhage. It is hypothesized that the focal areas of hsp70 induction may reflect ischemic injury due to vasospasm produced by lysed blood and/or injury mediated by direct toxic effects of the lysed blood. The hsp70 induction and DNA nick end-labeling in the same regions suggests that lysed blood produces a spectrum of injury from HSP70 protein-labeled, reversibly injured cells to dead cells with fragmented DNA. Induction of the hsp70 stress gene and DNA nick end-labeling may be useful for evaluating the causes of injury, the spectrum of injury, and potential pharmacological therapies in experimental models of subarachnoid hemorrhage.


Key Words: cerebral ischemia, focal • heat-shock proteins • subarachnoid hemorrhage • vasospasm


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Spontaneous SAH in humans and experimental SAH in animals increase intracranial pressure and reduce CPP.1 2 3 4 5 In animal models, even brief periods of intracranial hypertension and reduced CPP after SAH lead to perfusion defects throughout the brain.1 In baboons the decreases in CPP correlate with a reduction in CBF and diffuse brain edema as early as 2 hours after SAH.3 Recurrent SAH in humans may produce intracranial pressures that reduce CPP and CBF to zero.2 5 Primary and recurrent SAH can therefore rapidly decrease CPP and CBF to produce acute cerebral ischemia.6 7 8

SAH can also be complicated by delayed ischemic deficits that appear to result from vasospasm.9 Although CBF can be reduced as early as 15 minutes after experimental SAH,10 delayed decreases of CBF can occur for several weeks and produce infarcts.11 In addi-tion, the hemoglobin and catalytically active iron released after SAH can lead to lipid peroxidation and DNA degradation12 13 14 and may contribute to the delayed cellular damage. Although human cases and animal models of SAH repeatedly demonstrate vasospasm, there have not been reproducible methods for assessing the cellular injury associated with experimental SAH.

HSP70 is a heat-shock protein induced by many stresses including hyperthermia, seizures, and ischemia.15 16 17 18 19 20 21 22 23 24 25 HSP70 protein induction is a sensitive marker of neuronal injury.16 In the setting of focal ischemia, HSP70 is expressed in injured cells before any changes seen on H&E staining.20 21 In global17 25 and focal16 18 19 20 ischemia, HSP70 is expressed in injured cells in a cellular and regional hierarchy. Injured neurons express HSP70 earlier than glia and endothelium. After global ischemia, the CA1 neurons of hippocampus express HSP70 earlier than the CA3 neurons, which are more resistant to ischemia.17 Since little hsp70 mRNA and HSP70 protein are expressed in normal brain, the time course and topography of cellular injury can be followed by examining induction of the hsp70 gene.18 21 22

Therefore, we studied hsp70 mRNA and HSP70 protein induction in the brain after experimental SAH in rats. We also searched for evidence of cell death using a new technique to detect dead cells that have DNA fragmentation. Saline, oxyhemoglobin, BSA, whole blood, and lysed blood, a known spasmogen,26 were injected into the cisterna magna. The time course and topography of injury were then studied.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Experimental Model of SAH
All procedures were approved by our accredited animal care committee. Experimental SAH was produced in female Sprague-Dawley rats (n=72) weighing 200 to 250 g (Simonsen Labs, Gilroy, Calif) using the technique of Solomon et al.10 Rats were allowed food and water ad libitum before as well as after surgery. They were anesthetized with intraperitoneal injections of ketamine (80 mg/kg) and xylazine (8 mg/kg). To control for N-methyl-D-aspartate antagonist effects of ketamine, four rats injected with lysed blood were anesthetized with endotracheal anesthesia containing a mixture of 68.5% N2O, 30% O2, and 1.5% isofluorane for comparison. In these animals, blood pressure and blood gases were monitored, and pH, PO2, PCO2, and blood pressure were all maintained in the normal range.

After cannulation of the left femoral artery, rats were placed in a stereotaxic head holder (Kopf Instruments) with the head in 30° of flexion. A midline incision was made from the middle of the calvarium to the lower cervical spine. The occipital bone was cleared of muscular attachments by sharp dissection. Under a surgical microscope, dissection was carried down to the atlanto-occipital membrane with the use of microforceps. The membrane was cleared of connective tissue and divided to expose the arachnoid of the cisterna magna.

Animals (n=72) were randomly assigned to six experimental groups. The first group received a sham operation that consisted of needle puncture of the arachnoid without injection. The second group had 0.15 mL of saline injected into the cisterna magna. The third group had 0.15 mL of 1% BSA (Sigma Chemical Co) injected into the cisterna magna. The BSA was heated at 100°C for 1 hour, followed by freezing for 10 minutes and then thawing to denature the proteins. The fourth group was injected with 0.15 mL of autologous arterial blood. The fifth group had 0.15 mL (7.5 mg) of chromatographically purified oxyhemoglobin injected (prepared in our laboratory). The sixth group had 0.15 mL of lysed autologous arterial blood injected into the cisterna magna. The arterial blood was lysed by freezing in dry ice for 10 minutes, followed by thawing. In animals in which blood was withdrawn from the arterial line, an equal amount of normal saline was replaced.

A 1-mL syringe with a 30-gauge needle was lowered toward the cisterna magna at an angle of 60° from the horizontal under high magnification. A given substance was injected over 60 seconds. The needle was withdrawn after a few minutes' delay to allow cerebrospinal fluid circulation before any leakage from the puncture site. A piece of absorbable gelatin sponge (Gelfoam; Upjohn) was placed over the opening in the arachnoid. The skin was closed with 4-0 silk suture. The rats were given 5 mL of normal saline intraperitoneally and subcutaneously to prevent dehydration before recovery from anesthesia. Brain temperature was not controlled in these experiments; however, animals were placed on a heating pad to maintain body temperature at 37°C. Although animals were not routinely monitored physiologically, the mean arterial blood pressure (111±6 mm Hg), pH (7.39±0.05), PO2 (137±28 mm Hg), and PCO2 (36.7±7.4 mm Hg) were normal after subarachnoid injections of lysed blood in a small group rats (n=4) receiving endotracheal anesthesia and in which these parameters were measured after subarachnoid injections of blood.

Immunocytochemistry
The animals in the sham (n=2), saline (n=6), BSA (n=3), and oxyhemoglobin (n=5) groups were killed at 24 hours. The animals in the whole blood group were killed at 24 (n=4), 48 (n=2), 72 (n=2), and 96 hours (n=4). The animals in the lysed blood group were killed at 6 (n=2), 24 (n=8), 48 (n=2), 72 (n=2), and 96 hours (n=2). The rats were anesthetized with intraperitoneal ketamine (80 mg/kg) and perfused with the use of a cardiac catheter with 100 mL of 154 mmol/L (0.9%) saline followed by 400 mL of 4% paraformaldehyde in PB. Brains were postfixed in 4% paraformaldehyde for 1 to 3 hours, and 100-µm-thick coronal sections were cut on a Vibratome and placed in PB.

Immunocytochemistry was performed with the use of a mouse monoclonal antibody to HSP70 (C92, Amersham). The C92 antibody produces one band on Western blots of heat-shocked HeLa cells27 and two bands from ischemic brain25 and recognizes the protein expressed from the rat hsp70 cDNA.28 Immunocytochemistry was performed with the use of the avidin-biotin-horseradish peroxidase technique (Elite Vectastain, Vector Labs). Sections were placed in PB containing 2% sheep serum/0.1% BSA/0.2% Triton-X (Sigma Chemical Co) (PB-SS) for 2 hours at room temperature. This was followed by a 48-hour incubation at 4°C with the HSP70 monoclonal antibody diluted to 1:4000 in PB-SS. After three 5-minute washes in PB, sections were incubated with a biotinylated sheep anti-mouse IgG antibody (Amersham Life Sciences) at a dilution of 1:200 in PB-SS for 2 hours. All sections were washed again three times for 5 minutes each with PB, placed in avidin-horseradish peroxidase solution for 3 hours, washed in PB three times for 5 minutes each, and reacted for horseradish peroxidase with 0.56 mmol/L diaminobenzidine (0.015% in PB; Sigma Chemical Co) and 0.001% H2O2. Sections were dehydrated and coverslipped with the use of DePex (Biomedical Specialties). Alternate control sections were incubated without primary antibody and demonstrated no staining.

After immunocytochemistry, representative sections were counterstained with H&E (Anatech). Briefly, sections were dehydrated, delipidized, rehydrated, and counterstained with H&E. Sections were then dehydrated and coverslipped.

In Situ Hybridization
For in situ hybridization, one group of animals was killed at 6 hours after sham operations (n=2) or injections of saline (n=3), whole blood (n=3), or lysed blood (n=6). A second group of animals was killed at 24 hours after sham operations (n=2) or injections of saline (n=3), whole blood (n=3), or lysed blood (n=4). Rats were narcotized with CO2 and decapitated. Brains were removed and frozen in 2-methylbutane at -30°C. Sections (20 µm) were cut on a cryostat at -20°C, collected on precleaned slides, and processed for in situ hybridization according to the method of Schalling et al29 with modifications. The probe used in these studies was a synthetic oligonucleotide that corresponds to highly conserved amino acids (122-129) of the human hsp70 sequence.30 This probe detects the inducible hsp70 gene but not the constitutive hsc73 gene31 and detects little hsp70 mRNA in most normal rodent cells.22 28 32

The oligonucleotide probe was labeled with 35S-dATP (DuPont-NEN Res Products) with the use of TdT (DuPont-NEN). Sections were air dried and hybridized at 42°C for 18 hours in hybridization cocktail. Each 1 mL of cocktail contained 500 µL formamide, 200 µL of 4x SSC, 2 µL of 100x Denhardt's, 5 µL of 20% sarcosyl, 10 µL of 0.2 mol/L PB, 100 mg dextran sulfate, 0.5 mg salmon sperm DNA, 0.12 mmol dithiothreitol, and 1x107 cpm/mL of the labeled probe. Sections were then washed four times for 15 minutes each in 1x SSC at 55°C and thereafter left to cool for 1 to 3 hours at room temperature. Sections were then dipped in distilled water, 75% ethanol, and 90% ethanol and air dried at room temperature. They were covered with Kodak SB5 film, exposed for 1 to 2 weeks, and developed with Kodak GBX developer.

DNA Nick End-Labeling
Visualization of dead cells was performed with the use of modifications of the TdT dUTP-biotin nick end-labeling technique (TUNEL) described by Gold et al.33 Alternate sections of animals used for in situ hybridization were used; the animals had been killed at 24 hours after either saline (n=2), whole blood (n=3), or lysed blood (n=4) injection. Sections were prepared as described for in situ hybridization. They were fixed for 10 minutes with the use of 4% paraformaldehyde in 0.1 mol/L PBS, pH 7.4. Sections were placed in PBS for 15 minutes (twice), 0.3% Triton X-100 in PBS for 30 minutes, and PBS for 15 minutes (twice). TdT buffer (Gibco BRL) was then applied to the sections for 15 minutes. Excess buffer was removed, and a mixture of TdT (Gibco BRL) (300 U/mL) and biotin 14-dATP (40 mmol/L) in 1x TdT buffer was applied to the sections that were incubated at 37°C for 60 minutes. After the incubation, sections were washed in 2x SSC (150 mmol/L sodium chloride, 15 mmol/L sodium citrate, pH 7.4) for 15 minutes (twice) and 2% BSA in PBS for 15 minutes (twice). Avidin-horseradish peroxidase solution (Elite Vectastain) was applied to the sections for 30 minutes; they were then washed in sodium acetate solution (0.175 mol/L) for 15 minutes (twice). Staining was visualized with the use of 0.56 mmol/L diaminobenzidine (0.015%) and 30 mmol/L nickel sulfate (1%) in 0.175 mol/L sodium acetate solution. Slides were washed in distilled water, dried, and coverslipped with Depex. The positive DNA nick end-labeled cells may have died by either apoptosis or necrosis. In either case, the DNA nick end-labeling indicates that the DNA in these cells has been cut into many pieces and therefore that the cells are dead or will die. H&E staining was used to counterstain sections that were reacted for DNA nick end-labeling.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
HSP70 Protein Induction in Forebrain
There was little HSP70 immunostaining in the forebrain of sham subjects or those injected with saline, oxyhemoglobin, BSA, and whole blood (Fig. 1ADown) at any time point after injection. At 24 hours after injection of lysed blood, however, HSP70 immunoreactivity was observed in 8 of 8 experimental subjects in focal areas that were distributed throughout the forebrain but with a preponderance at the base (Fig 1BDown and 1CDown). Focal areas were also seen in dorsal and lateral neocortex, septal nuclei, striatum, and white matter tracts (Fig 1DDown). HSP70 was not induced in hippocampus or thalamus. Regions of focal induction were seen most often at the base of the brain (Fig 1BDown) adjacent to where the lenticulostriate arteries arose and where the injected blood likely collected in high concentrations in the basal cisterns.




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  		Figure 1. HSP70 immunocytochemistry of rat forebrain 24 hours after injection of saline into the cisterna magna (A) and 1 day (B, C, D) and 4 days (E) after cisterna magna injections of lysed blood. HSP70 protein was induced mainly in glia (C through E) in patches located near the base of the brain in hypothalamus (B), basal cortex (solid arrow in B, C, E) and anterior commissure (arrowhead in B, D) after injection of lysed blood. C and D represent higher magnification views of the regions denoted in B by the solid arrow and arrowhead, respectively. Bar=500 µm for A and B and 100 µm for C through E.

HSP70 immunoreactivity in the basal forebrain revealed dense staining in cells with multiple processes resembling microglia (Fig 1CUp) and occasionally astrocytes. Neuronal induction of HSP70 was observed less often, and then mostly in dorsolateral cortex (not shown). On occasion, the dense glial staining would surround a pale area of stained cells resembling shrunken neurons. In the white matter tracts, HSP70 immunostaining occurred in cells that occasionally resembled astrocytes (presumably type 2) or oligodendrocytes (Fig 1DUp). On H&E-counterstained sections, a normal histological appearance was observed in many areas of HSP70 immunostaining. In certain regions of HSP70 immunostaining, a characteristic morphology was observed that consisted of a rim of HSP70 immunostaining surrounding a core of scant HSP70 immunoreactivity. H&E counterstaining in these regions demonstrated normal histology within the rim of HSP70 immunoreactivity with evidence of coagulative necrosis in the core.

HSP70 Protein Induction in the Cerebellum
HSP70 immunoreactive cells were not observed in the brain stem or cerebellum of sham-operated animals or in animals injected with saline, oxyhemoglobin, or whole blood (Fig 2ADown) at any time point. After lysed blood injection, however, HSP70 immunoreactivity was apparent in the cerebellum beginning at 6 hours (2 of 2 subjects) and continuing for at least 24 hours in all (8 of 8) subjects (Fig 2BDown and 2CDown). At 6 hours, HSP70 induction was observed in neurons presumed to be stellate cells, occasional Purkinje cells, and granule cell neurons. By 24 hours, HSP70 protein induction occurred mainly in molecular layer Bergmann glia (Fig 2BDown and 2CDown). HSP70 immunoreactivity was detected in white matter of cerebellum and brain stem in half of the subjects (4 of 8).



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  		Figure 2. HSP70 immunocytochemistry of rat cerebellum 24 hours after injection of saline into the cisterna magna (A) and 1 day (B, C) and 4 days (D) after cisterna magna injections of lysed blood. HSP70 protein was induced throughout the cerebellar gray matter in the molecular (open arrow in B, large arrowhead in C) and granule cell layers (solid arrow in B, open arrow in C) and was localized to Bergmann glia (open arrow in B, large arrowhead in C), granule cell neurons (open arrow in C), Purkinje cell neurons (not shown), scattered stellate neurons in the molecular layer (small arrowhead in C), and other unidentified cells. Staining was decreased at 4 days (D) compared with 1 day (B, C) after injections. C represents a magnified view of B. Bar=500 µm for A and B and 100 µm for C and D.

Most HSP70 immunoreactive cells in the molecular layer contained multiple processes extending from the Purkinje cell layer to the subarachnoid space indicative of Bergmann glia (Fig 2CUp). The second cell type was smaller with few processes and was contained entirely within the molecular layer and resembled stellate cells (Fig 2CUp). In some of the regions where HSP70 was induced in the molecular layer, HSP70 immunoreactivity was also seen in adjacent granule cells (Fig 2CUp, top). Occasional HSP70 immunoreactive Purkinje cells were also observed. The HSP70 immunoreactive cells in the white matter in cerebellum and brain stem resembled those seen in the forebrain and were most likely astrocytes or microglia. H&E counterstaining of these sections demonstrated a normal histological appearance in the molecular, Purkinje cell, and granule cell layers of the cerebellum. Coagulative necrosis was not observed on H&E counterstaining of brain stem and cerebellum.

Time Course of HSP70 Protein Induction and Resolution
At 2, 3, and 4 days after whole blood injections, HSP70 immunoreactivity was never induced in the forebrain or hindbrain (not shown). At 2, 3, and 4 days after lysed blood injections, HSP70 protein was induced in forebrain (Fig 1EUp) and cerebellum (Fig 2DUp) in patterns similar to those observed in subjects killed at 1 day (Figs 1B through 1D and 2B and 2C). The HSP70 immunoreactive regions were fewer, smaller, and less dense at 3 to 4 days compared with 1 day after lysed blood injections.

hsp70 mRNA Induction in the Forebrain and Brain Stem
There was no induction of hsp70 mRNA in either the forebrain (Fig 3ADown) or cerebellum (Fig 3DDown) in sham, saline (Fig 3ADown and 3DDown), and whole blood groups at either 6 hours (Fig 3ADown and 3DDown) or 24 hours after the injections. After injections of lysed blood, hsp70 mRNA was induced in the forebrain in 3 of 6 subjects at 6 hours (Fig 3BDown) and 3 of 4 subjects at 24 hours (Fig 3CDown). At 6 hours, hsp70 mRNA was seen in focal areas in the basal forebrain of 2 subjects (Fig 3BDown). However, 1 subject had diffuse hsp70 mRNA induction in the neocortex bilaterally at 6 hours (not shown). At 24 hours, hsp70 mRNA was also seen in focal areas in the basal forebrain of all subjects (Fig 3CDown).



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  		Figure 3. hsp70 in situ hybridization of forebrain (A through C) and brain stem/cerebellum (D through F) performed 24 hours after injections of saline into the cisterna magna (A, D) and 6 hours (B, E) and 24 hours (C, F) after injections of lysed blood into cisterna magna. hsp70 mRNA was induced in small patches at the base of the forebrain at 6 hours (solid arrow in B) and 24 hours (solid arrow in C) after the lysed blood injections. hsp70 mRNA was also induced in the cerebellar vermis and cerebellar hemispheres at 6 hours (E) and 24 hours (F) after the injections of lysed blood. Bar=2 mm.

In the lysed blood group, hsp70 mRNA was induced in the cerebellum of 5 of 6 subjects at 6 hours (Fig 3EUp) and 3 of 4 subjects at 24 hours (Fig 3FUp). hsp70 mRNA induction in cerebellum was distributed in the molecular and granular layers. Little hsp70 mRNA was induced in the cerebellar white matter (Fig 3EUp and 3FUp). hsp70 mRNA induction in cerebellum appeared to decrease over time from 6 to 24 hours (Fig 3EUp and 3FUp).

Cell Death in Focal Regions After Injections of Lysed Blood
There was no DNA nick end-labeling detected in the forebrain of the animals injected with saline (Fig 4ADown) or whole blood (not shown). However, DNA nick end-labeling was detected in the forebrain of 3 of 4 subjects injected 24 hours previously with lysed blood (Fig 4BDown). The DNA nick end-labeled cells were found in focal areas at the base of the forebrain (Fig 4BDown). The focal areas of DNA nick end-labeled cells correlated exactly with the focal regions of hsp70 mRNA induction on the in situ hybridization studies on alternate sections. Furthermore, the 1 subject that had no DNA nick-end labeling in the basal forebrain also did not show any hsp70 mRNA induction in the basal forebrain.



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  		Figure 4. DNA nick end-labeling of sections from the basal forebrain (A, B) of animals 24 hours after the injection of either saline (A) or lysed blood (B) into the cisterna magna. Note the absence of DNA nick end-labeled cells in the basal forebrain of a saline injected subject (A) compared with numerous labeled cells in the basal forebrain of a subject injected with lysed blood (B). Injection of whole blood into the cisterna magna did not demonstrate DNA nick end-labeling (not shown). Bar=25 µm.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
Regional Distribution of HSP70 Protein and hsp70 mRNA Induction
After lysed but not whole blood injections into the cisterna magna, hsp70 mRNA and HSP70 protein were induced in focal areas in the forebrain. H&E counterstaining of these sections was usually normal in those regions with HSP70 immunoreactivity but occasionally demonstrated focal areas of coagulative necrosis. Morphologically and histologically, the regions of HSP70 immunoreactivity resembled those described by Sharp et al23 24 in the striatum after global ischemia in the rat. In that study, the mechanism of HSP70 induction was proposed to be microvascular ischemia from delayed hypoperfusion. Since lysed blood is a known spasmogen,26 34 the multifocal regions observed in our study may have been the result of ischemia from either decreased CPP or vasospasm. The distribution of focal HSP70 induction in our study appeared mostly in territories supplied by small perforating arteries, including the lenticulostriates, and occurred in regions adjacent to where high concentrations of lysed blood would be expected in the basal cisterns of the subarachnoid space. This pattern of HSP70 immunoreactivity might suggest that HSP70 induction could be used as a possible marker for vasospasm. The H&E counterstaining performed in this study suggested that HSP70 immunostaining is a more sensitive indicator of cellular injury than conventional histology after SAH, a finding consistent with that observed in focal ischemia.20 Further studies will be required to determine whether the areas of HSP70 induction correlate with areas of decreased perfusion.

In cerebellum, HSP70 protein and mRNA were induced in a broad distribution in neurons and glia in the molecular layer and granule cell neurons. H&E counterstaining of these same sections did not reveal any evidence of coagulative necrosis or neuronal damage and suggested that these cells were injured but not dead. This pattern of cerebellar injury was suggestive of a diffuse process. Since lysed blood was injected in the cisterna magna in the areas adjacent to cerebellum, diffuse vasospasm in the circumferential arteries may have produced broad ischemic territories in the cerebellar hemispheres and vermis and resulted in this significant HSP70 induction. An alternative hypothesis is that the lysed blood produced a direct toxic injury on cells in the cerebellum, resulting in diffuse HSP70 induction. It is also possible that some combination of vasospasm and direct toxic effects of lysed blood account for HSP70 induction in both forebrain and cerebellum.

Dead Cells Occur in the Same Forebrain Regions Where HSP70 Protein Is Induced
The data show that cells die within the focal areas of HSP70 induction and that these cells may die in part by apoptosis since they have evidence of DNA nick end-labeling.33 35 Because a number of studies have suggested that apoptosis occurs after both focal and global ischemia in the brain,36 37 38 the apoptotic cell death in forebrain after SAH is consistent with ischemic injury to these cells. The DNA nick end-labeled cells are unequivocally dead because their DNA has been cleaved into small "nucleosomal-sized" pieces. This demonstrates that cisternal injections of lysed blood produce a spectrum of cellular injury in the focal regions: from dead, DNA nick end-labeled cells to HSP70 immunostained cells that are generally believed to survive the injury.21 These data are also important for demonstrating that the focal areas represent not only stress gene induction but also areas of injury sufficient to kill cells.

Mechanisms of HSP70 Induction: Lysed Versus Whole Blood
Whatever the explanation for the HSP70 induction in forebrain and cerebellum and the DNA fragmentation and cell death in forebrain, an important finding of this study is that lysed but not whole blood or oxyhemoglobin produced cell death with DNA fragmentation and induced HSP70. After injection of whole blood into the cisterna magna of rats, a biphasic pattern of arterial narrowing has been described.39 The first phase occurs at 10 minutes, while the second phase is observed at 48 hours. Decreases in CBF have also been demonstrated after injection of whole blood into the cisterna magna of rats.10 However, despite reductions in CBF, single injections of whole blood into the cisterna magna may not produce enough arterial narrowing and ischemia to induce HSP70. After a single injection of whole blood into the cisterna magna, blood is rapidly cleared from the subarachnoid space.34 A single injection of whole blood may not produce a consistent model of vasospasm.34 In canine models, two injections of whole blood 72 hours apart provide a greater degree of arterial narrowing than that observed after one injection.40 When SAH is induced in the rat by basilar arterial puncture, a larger, more prolonged reduction in CBF is observed than that demonstrated after injection of whole blood into the cisterna magna.11 Furthermore, studies in our laboratory have demonstrated HSP70 induction throughout rat forebrain after SAH induced by endovascular arterial perforation (P. Matz, S. Sunderson, F. Sharp, P. Weinstein, unpublished data, 1995). These data demonstrate that whole blood can induce the HSP70 protein if present in high concentrations. In this context it is important to note that in the present model whole blood also did not produce focal brain injury at any time between 6 hours and 4 days and that focal injury only occurred in the lysed blood group. It seems likely that the major difference between the lysed and whole blood group after a single injection into the cisterna magna is the greater concentration of one or more substances released into the subarachnoid space from lysed compared with whole blood. This would suggest that the chances of cellular injury are much greater with greater concentrations of blood that lyse at high rates.

Lysed blood is a more potent spasmogen than whole blood. In canine models, lysed blood injection into the cisterna magna produces rapid, severe arterial narrowing that persists for several days.26 This type of persistent, severe arterial narrowing in rats may produce ischemia and cause HSP70 induction. Oxyhemoglobin is also a potent spasmogen.34 Even though oxyhemoglobin has been shown to produce vasospasm in animal models, it is thought that a concentration of 10-3 mol/L is required to produce significant vasospasm in vivo.34 In our study, 7.5 mg of chromatographically purified hemoglobin A0 was injected into the subarachnoid space in the oxyhemoglobin group. However, it was estimated that the total amount of hemoglobin A0 in the 0.15 mL of lysed blood was at least twice as great. This additional amount of oxyhemoglobin in the lysed blood group may have been great enough to produce vasospasm in the forebrain and cerebellum. Exactly what the toxic and/or vasospastic substances are in the lysed blood cannot be inferred from the present studies but has been the subject of many studies.8 34 41 The current data suggest that one reason for "delayed vasospasm" in human cases of SAH could be related to lysis of blood in the subarachnoid space and accumulation of sufficient concentrations of the offending compounds.

The possible mechanisms of hsp70 mRNA and HSP70 protein induction by ischemia are reviewed elsewhere21 but involve activation of heat-shock factors by the presence of denatured proteins within cells. Heat-shock factors then bind to the heat-shock elements in the promoters of heat-shock genes and initiate transcription of hsp70 mRNA.21 It is unlikely that denatured proteins in the subarachnoid space, per se, account for the hsp70 induction observed after lysed blood injections since injections of denatured BSA did not induce HSP70 in this study.

Time Course of HSP70 Protein and hsp70 mRNA Induction
Induction of hsp70 mRNA is a very early marker for cellular injury, being induced as early as 1 to 4 hours after ischemia.18 21 In forebrain and cerebellum hsp70 mRNA was induced at 6 hours after injection of lysed blood and persisted for at least 24 hours. The time course of hsp70 mRNA induction suggested that cellular injury in forebrain and cerebellum occurred at least by 6 hours and may have occurred even earlier after subarachnoid injections of lysed blood. The progression of hsp70 mRNA in forebrain in the first 24 hours after injections was similar to that seen after focal cerebral ischemia.18 In both forebrain and cerebellum, HSP70 protein was induced and peaked at 24 hours and then decreased by 96 hours after injection of lysed blood, which resembled the time course in focal forebrain ischemia.18 The similarities to focal ischemia in the progression of forebrain hsp70 mRNA and HSP70 protein induction after experimental SAH are compatible with our hypothesis that cellular injury occurs from ischemia as a result of vasospasm caused by the lysed blood.26 34

Cellular Induction of HSP70 Protein
The cellular induction of HSP70 in forebrain after SAH differed from that seen after focal ischemia18 since HSP70 was induced mainly in glia rather than mainly in neurons. However, after global ischemia some of the areas in which HSP70 was induced demonstrated mainly glial staining.23 In addition, it is possible that the injury produced by the experimental SAH is a combination of ischemia, produced by decreased CPP and/or vasospasm, and toxic effects of blood on glial cells. Glia may play a primary role in metabolizing blood products in the subarachnoid space and in sequestering catalytically active iron and hence may become sensitive to cellular injury. In addition, neurons destined to die in the setting of ischemia may not make hsp70 mRNA, and even if they do they may not translate the hsp70 mRNA into protein.19 21

In cerebellum, the pattern of HSP70 protein induction after SAH was also unique in that HSP70 was induced in multiple cell types, including granule cells, stellate cells, Purkinje cells, and Bergmann glia. Moreover, neuronal HSP70 induction preceded that in the glia. This progression suggested that neurons were more sensitive to SAH injury and were injured the earliest. Hyperthermia has previously been shown to induce HSP70 protein in granule cell neurons, astrocytes, and endothelial cells32 but not in Bergmann glia. After sublethal global and focal cerebral ischemia, HSP70 is induced in a cellular hierarchy with HSP70 induction in neurons occurring at lower ischemic thresholds than cells more resistant to ischemia like glia and endothelium.16 17 18 19 20 After cisterna magna injection, the concentration of lysed blood was maximal in the subarachnoid space around the base of the cerebellum. This may have led to diffuse vasospasm of the circumferential arteries and early neuronal injury from ischemia. HSP70 protein may be induced in Bergmann glia at later times because glia are more resistant to ischemic injury.

HSP70 Induction and Cerebral Protection After SAH
In addition to delineating the distribution and time course of cellular injury, HSP70 protein induction also may play a role in protecting cells from further injury.42 43 44 45 46 47 Induction of HSP70 protein, which binds to denaturing proteins and prevents them from becoming further denatured, has been shown to protect cultured cells, brain, and retina against a variety of injuries.21 The possibility that prior induction of HSP70 may protect the brain against injury produced by SAH has not been examined. It is possible that the induction of hsp70 and other stress genes caused by SAH might temporarily protect the brain during the first week or so, perhaps helping to explain the time course of the delayed ischemic deficits in patients after SAH.6 8

New Methods for Assessing Injury in Experimental SAH
The induction of hsp70 mRNA and HSP70 protein and the presence of DNA nick end-labeled cells allowed us to ascertain the time course, regional distribution, and cellular hierarchy of injury after cisterna magna injections of lysed blood. The DNA nick end-labeling provides an unequivocal method for detecting dead cells in small regions of the brain. Since induction of the hsp70 gene is a more sensitive indicator of cell injury than conventional histological techniques,20 21 we propose that this will provide a new and sensitive means of assessing the cellular injury caused by SAH in animal models.


*    Selected Abbreviations and Acronyms
 
BSA = bovine serum albumin
CBF = cerebral blood flow
CPP = cerebral perfusion pressure
H&E = hematoxylin and eosin
PB = phosphate buffer
SAH = subarachnoid hemorrhage
TdT = terminal deoxynucleotidyl transferase


*    Acknowledgments
 
This study was supported by National Institutes of Health grants NS-28167, NS-14543, and HL-53040 (Dr Sharp), the Merit Review Program of the Department of Veterans Affairs (Drs Sharp and Weinstein), and the Finnish Academy of Sciences and Finnish Neurology Association (Dr Honkaniemi).

Received July 21, 1995; revision received October 18, 1995; accepted November 27, 1995.


*    References
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 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
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*References
 

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