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Am J Physiol Regul Integr Comp Physiol 292: R403-R414, 2007. First published September 14, 2006; doi:10.1152/ajpregu.00403.2006
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Increased expression of HIF-1{alpha}, nNOS, and VEGF in the cerebral cortex of anemic rats

Anya T. McLaren,1,2 Philip A. Marsden,3 C. David Mazer,1,2 Andrew J. Baker,1 Duncan J. Stewart,4 Albert K. Y. Tsui,1,2 Xiaomao Li,1,5 Yeni Yucel,6,7 Malcolm Robb,4 Shelley R. Boyd,7 Elaine Liu,1 Julie Yu,1 and Gregory M. T. Hare1,2

1Department of Anesthesia, Cara Phelan Trauma Research Centre, 3Division of Nephrology, 4Division of Cardiology, 6Department of Laboratory Medicine and Pathobiology, 7Department of Opthalmology, University of Toronto, St. Michael's Hospital, Toronto, Ontario; 2Department of Physiology, University of Toronto, Toronto, Ontario; and 5Biotechnology Centre for Applied Research and Training, Seneca College, Toronto, Ontario, Canada

Submitted 8 June 2006 ; accepted in final form 30 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study tested the hypothesis that specific hypoxic molecules, including hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), neuronal nitric oxide synthase (nNOS), and vascular endothelial growth factor (VEGF), are upregulated within the cerebral cortex of acutely anemic rats. Isoflurane-anesthetized rats underwent acute hemodilution by exchanging 50% of their blood volume with pentastarch. Following hemodilution, mean arterial pressure and arterial PaO2 values did not differ between control and anemic rats while the hemoglobin concentration decreased to 57 ± 2 g/l. In anemic rats, cerebral cortical HIF-1{alpha} protein levels were increased, relative to controls (1.7 ± 0.5-fold, P < 0.05). This increase was associated with an increase in mRNA levels for VEGF, erythropoietin, CXCR4, iNOS, and nNOS (P < 0.05 for all), but not endothelial NOS. Cerebral cortical nNOS and VEGF protein levels were increased in anemic rats, relative to controls (2.0 ± 0.2- and 1.5 ± 0.4-fold, respectively, P < 0.05 for both). Immunohistochemistry demonstrated increased HIF-1{alpha} and VEGF staining in perivascular regions of the anemic cerebral cortex and an increase in the number of nNOS-positive cerebral cortical cells (3.2 ± 1.0-fold, P < 0.001). The nNOS-positive cells costained with the neuronal marker, Neu-N, but not with the astrocytic marker glial fibrillary acidic protein (GFAP). These nNOS-positive neurons frequently sent axonal projections toward cerebral blood vessels. Conversely, VEGF immunostaining colocalized with both neuronal (NeuN) and astrocytic markers (GFAP). In conclusion, acute normotensive, normoxemic hemodilution increased the levels of HIF-1{alpha} protein and mRNA for HIF-1-responsive molecules. nNOS and VEGF protein levels were also increased within the cerebral cortex of anemic rats at clinically relevant hemoglobin concentrations.

hemodilution; cerebral hypoxia

ACUTE HEMODILUTION AND ANEMIA have been associated with increased perioperative mortality (6, 12), cognitive and neurophysiological dysfunction (51), and increased neurological injury in surgical patients (18, 25). The mechanisms underlying these poor outcomes in anemic patients are currently undefined. One experimental study suggests that inadequate cerebral tissue oxygen supply may contribute to cerebral injury following acute hemodilution (21). However, little is understood about other potential mechanisms of organ injury. As with other paradigms of cerebral injury, regulatory mechanisms are likely activated to protect the brain during acute anemia. However, our understanding of which mechanisms are activated and how they operate is limited.

General regulatory responses to acute anemia include a reduction in overall systemic metabolic rate (30), increased cardiac output and blood flow to vital organs (43, 47), and promotion of angiogenesis (38). With respect to the brain, a preferential increase in cerebral blood flow (CBF) is observed, relative to other less vital organs, thereby preserving cerebral tissue oxygen delivery during severe hemodilution (43, 47). This adaptive response occurs by active cerebral vasodilation, which is partially mediated by nitric oxide synthase (NOS) activity (20, 23). However, at very low hemoglobin concentrations, these regulatory mechanisms are overwhelmed and cerebral tissue hypoxia occurs (47).

Hypoxia is known to activate a number of regulatory responses within the cerebral cortex, including an increase in expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) (2), neuronal NOS (nNOS) (7, 17, 50), vascular endothelial growth factor (VEGF) (2), erythropoietin (EPO), inducible NOS (iNOS), and chemokine receptors (CXCR4) (2, 31, 41). Each of these responses may support adaptive mechanisms, which protect the brain from hypoxic injury through adaptation of cerebral metabolism (1, 30), regulation of CBF (8, 23), promotion of angiogenesis (34, 53), and cytoprotection (2, 24).

The functional interactions between HIF-1{alpha}, nNOS, and VEGF can differ depending on the level of tissue oxygen tension (3). Under hypoxic conditions, increased NO can reduce cerebral HIF-1{alpha} and VEGF expression (22, 44). However, under normoxic conditions, increased NO may stabilize HIF-1{alpha} levels leading to transcription of HIF-1-responsive elements, including VEGF (48). These mechanisms may be important in anemia since a recent experimental study has demonstrated evidence of increased cerebral cortical nNOS gene expression within the cerebral cortex of anemic rats (20). The current study tests the hypothesis that acute hemodilutional anemia will result in activation of specific adaptive regulatory mechanisms within the cerebral cortex, including HIF-1{alpha}, nNOS, and VEGF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Model

All animal protocols were approved by the Animal Care and Use Committee at St. Michael's Hospital in accordance with the requirements of Canadian Animal Care. Anesthesia was induced in male Sprague-Dawley rats (Charles River, St. Constant, PQ) with 3–4% isoflurane in 100% oxygen in an induction chamber and maintained with 1–2% isoflurane in 50% oxygen (Abbott, St. Laurent, PQ). Following intubation, ventilation was maintained with a pressure-controlled ventilator (Kent Scientific, Litchfield, CT). Incision sites were infiltrated with 2% lidocaine before cannulation of the tail artery and vein (24-gauge angiocath, BD Medical, Oakville, ON). Vascular access was utilized to measure mean arterial blood pressure (MAP), arterial blood gases (ABGs), and hemoglobin concentration by co-oximetry (Radiometer ALB 500 and OSM 3 London Scientific, London, ON) and to perform acute hemodilution. Ventilation was adjusted to achieve normocapnia and normoxia as determined by blood gas analysis. A heating pad and heating lamp were used to maintain rectal temperature near 37°C. MAP was recorded using a computerized data-acquisition system (DASYLab 5.6, Kent Scientific).

After baseline ABGs and hemoglobin concentrations were established, acute hemodilutional anemia was induced by simultaneously exchanging 30 ml/kg of arterial blood (50% of the estimated blood volume), withdrawn from the tail artery, with an equivalent volume of 10% pentastarch (Pentaspan, Bristol-Myers Squibb, Montreal, PQ) infused via the tail vein (n = 6 rats per time period). Volume exchange was performed over 10 min using a programmable "push-pull" pump as previously described (PHD 2000, Harvard Apparatus, Saint-Laurent, PQ) (20). Following completion of volume exchange, all parameters were recorded for an additional 20 min. Rats were then recovered until they were spontaneously breathing room air. Two groups of control animals were utilized. Initially, control animals underwent simultaneous exchange transfusion with whole blood collected from an anesthetized donor rat to control for the effect of volume exchange (early recovery experiments and HIF-1{alpha} protein determinations). For subsequent control experiments, rats underwent similar anesthesia and cannulation procedures but did not undergo hemodilution or volume exchange. Hypoxic positive control rats for nNOS and VEGF protein assays were placed in a hypoxic chamber (10% oxygen in nitrogen) for 6, 12, and 24 h (n = 6 rats per time period).

Experimental Protocol 1: Acute Recovery from Hemodilution

Effect of hemodilution on MAP and PaO2. A subgroup of control and anemic rats (n = 6 per group) had an additional femoral artery cannula inserted to monitor MAP during hemodilution. Following hemodilution, spontaneous ventilation was reestablished and animals were recovered breathing room air. The tail artery cannula was maintained to assess the hemoglobin concentration and ABG values in the immediate postoperative period. Control rats did not undergo hemodilution. Blood gas measurements were performed before, during, and after hemodilution. The final sample was obtained ~1 h after resumption of spontaneous ventilation on room air to determine whether hypoxemia was present. Rats were then killed with ketamine (100 mg/kg iv).

Experimental Protocol 2: Hemodilution and 6- to 24-h Recovery

Effect of hemodilution on cerebral HIF-1{alpha}, VEGF, and nNOS mRNA and protein. Different groups of rats underwent 50% hemodilution and then recovered for 6, 12, 18, and 24 h (n = 6 per group and time). At the time of death, rats were anesthetized with 2% isoflurane in an induction chamber, before blood sampling by cardiac puncture. Co-oximetry was used to assess the hemoglobin concentration. Anesthetized animals were then decapitated and brain tissue was harvested under sterile conditions. Control animals underwent autologous blood exchange (protocol 1) or did not undergo hemodilution (protocol 2).

Cerebral cortical RNA extraction and RT-PCR. Samples of cerebral cortex were flash-frozen in liquid nitrogen within 4 min of decapitation and stored at –80°C until RNA extraction was performed. Following mechanical disruption of tissue by homogenization, total RNA was extracted using standard techniques (RNeasy Mini Kit, Qiagen, Mississauga, ON). The OD 260/280 {lambda} was determined using 10 µl of sample and the remaining volume was divided into aliquots and frozen and stored in a –80°C freezer. Reverse transcription (RT) of 1–2 µg of total RNA to cDNA was performed using moloney-murine leukemia virus reverse transcriptase (M-MLV; Invitrogen, Burlington, ON) and random 6 bp primers (Invitrogen). A prepared M-MLV enzyme mixture (8 µl) consisting of 4 µl first-strand buffer, 2 µl of 0.1 M DTT, 1 µl 10 mM dNTP, and 1 µl M-MLV enzyme was added to each sample of cDNA and incubated at 37°C for 1 h and 30 min. Enzyme activity was neutralized by heating samples to 95°C for 2 min before initiation of the PCR reaction. Real-time quantitative PCR was performed utilizing SYBR Green fluorescent dye read by the ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Utilizing the described ABI methodology, we designed gene-specific primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), nNOS, eNOS, iNOS, CXCR4, HIF-1{alpha}, and VEGF (Table 1). Gene-specific PCR products were generated using DNA polymerase (Qiagen, Quantitech, SYBR Green RT-PCR kit no. 204243). Briefly, 40 PCR cycles were performed, including denaturing at 95°C for 15 s and annealing and extension for 1 min. The PCR product size limitation was between 96 to 342 bases. For GAPDH, nNOS, HIF-1{alpha}, and VEGF, the target transcript quantities were determined by measurement of the PCR product against a standard curve. The standard curves were generated using a dsDNA template constructed to match the sequence between the 5'-ends of the forward and reverse primers, relative to their cDNAs. The standard templates underwent five 10-fold serial dilutions to produce a five point standard curve. The standard curve values ranged from 100 to 0.01 pg for GAPDH. For the target genes nNOS, HIF-1{alpha}, and VEGF, standard curve values ranged from 1,000 to 0.1 fg. Quantitative PCR product quantities are reported in picograms or femtograms per 30 µl of reaction mixture. Such standard curves were not constructed for EPO, eNOS, iNOS, and CXCR4. Quantitation of eNOS, iNOS, CXCR4, and EPO mRNA levels was achieved by normalizing data to GAPDH using the delta delta Ct ({Delta}{Delta}Ct) method and reported as fold-change relative to the 6-h control group. Melting curve analysis was performed to verify the uniformity of all PCR products.


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  		Table 1. Primers utilized for real-time PCR

 
Cerebral cortical protein extraction and Western blot analysis. For measurement of HIF-1{alpha}, cerebral cortical tissue samples were mechanically homogenized (PowerGen 125, Fisher Scientific, Ottawa, ON, Canada) with ice-cold buffer (20 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 0.45 M NaCl) supplemented with 0.5 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and 0.2 mM DTT. The homogenates were centrifuged at 10,000 g at 4°C for 30 min, and supernatants were mixed with an equal volume of buffer with 40% vol/vol glycerol. For nNOS and VEGF, a similar procedure was undertaken with a homogenization buffer consisting of 10 mM HEPES, 0.1 mM EDTA, 1 mM dithioreitol, 1 mg/ml PMSF, 0.32 mM sucrose, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin A at pH 7.4. Protein assays were carried out using the Lowry method with bovine serum albumin (BSA) as a standard (Bio-Rad). Individual aliquots corresponding to 50 µg of total proteins were stored at –80°C before Western blot analysis.

Control, anemic, and hypoxic cerebral cortical samples (50 µg total protein in 10 µl) were subjected to 7.5% SDS-PAGE under reducing conditions (HIF-1{alpha}) or 10% SDS-PAGE under nonreducing conditions (nNOS, VEGF). Positive control samples consisted of hypoxic rat retinal lysate (HIF-1{alpha}), rat cerebellar lysate (nNOS, cat. no. 611463, BD Biosciences, Mississauga, ON, Canada), and mouse whole cell lysate (VEGF, NIH/3T3 cat. no. 2210, Santa Cruz Biotechnology, Santa Cruz, CA). Following electrophoresis, samples were then transferred to PVDF membranes (Bio-Rad) and incubated overnight at 4°C with primary antibodies for either HIF-1{alpha} (rabbit polyclonal anti-HIF, NB 100–497, Novus Biologicals, Littleton, CO), nNOS (monoclonal antibody, cat. no. 610309, BD Biosciences), VEGF (rabbit polyclonal antibody, cat. no. 507, Santa Cruz Biotechnology), or eNOS (monoclonal antibody, cat. no. 610296, BD Biosciences). After being washed, blots were incubated with an appropriate secondary antibody conjugated with horseradish peroxidase. Chemiluminescent detection reagents (ECL Western blotting system, Amersham Pharmacia Biotech, Baie d'Urfe, PC) were added followed by exposure to Kodak Bio-max film. Developed films were scanned with a Hewlett Packard Pro scanner. Optical densities of the positive protein bands were quantified using the Alpha Imager software (AlphaImager, 8-bit digital camera, Alpha Innotech, San Leandro, CA). Staining for beta-actin was performed to assess uniformity of protein loading and transfer. All data are normalized to the corresponding beta-actin band density and reported as a percentage increase over control values. HIF-1{alpha}, nNOS, and VEGF bands were identified at ~120, 155, and 46 kDa, respectively.

Immunohistochemistry and immunofluorescence. An additional four anemic and control rats were prepared and recovered for 18 h. At 18 h, rats were anesthetized (Nembutal, 50 mg/kg ip pentobarbital sodium) and killed by aortic perfusion with 4% paraformaldehyde in phosphate-buffered saline (PBS), and brain tissue was fixed overnight in 4% paraformaldehyde at 4°C. Following dehydration, tissue was placed in 100% ethanol and xylene (1:1 vol:vol) before being processed. Mounted tissue was cut into 10-µm sections using a microtome (RM 2135, Leica Microsystems, Richmond Hill, ON, Canada) and mounted onto glass slides. Paraffin sections were then dewaxed and blocked with 10% horse serum in 10% PBS for 60 min at room temperature. Immunohistochemical staining was performed by incubating slides overnight at 4°C with specific primary monoclonal antibodies (1:50 to 1:400 dilution) for HIF-1{alpha} (NB100–105, Novus Biologicals), nNOS (610308, BD Biosciences), and VEGF (AB1316, Cedarlane Laboratories, Hornby, ON). Slides were then incubated with a secondary biotinylated horse anti-mouse antibody (1:200 dilution; Vectastain kit, Vector Laboratories, PK-8800, Burlington, ON). An avidin/biotinylated enzyme complex and enzyme substrate (DAB) were added to visualize the immunoreactive material. Nuclear counterstaining was performed using hematoxylin. Quantitation of nNOS-positive cells was performed independently by two blinded individuals and reported as nNOS-positive cerebral cortical or basal ganglion cells per coronal section (n = 4). Negative staining controls were performed in the absence of primary monoclonal antibody.

Immunofluorescent staining was performed in the following combinations with indicated primary antibodies: 1) nNOS (Rabbit Polyclonal, 610310, BD Biosciences) and NeuN (Mouse, Monoclonal, MAB377, Chemicon International, Temecula, CA); 2) nNOS, GFAP (Mouse Monoclonal, C9205, GFAP-Cy3 conjugated, Sigma), and To-Pro-3 (642/661 Molecular Probes, Eugene, OR); 3) VEGF (rabbit polyclonal, SC-147 Santa Cruz Biotechnology) and NeuN; 4) VEGF, GFAP, and To-Pro-3, smooth muscle actin (Lab Vision, MS-113-PO clone 1A4, Fremont, CA) and nNOS; and 5) HIF-1{alpha} and VEGF. After application of primary antibodies, appropriate specific noncross-reacting fluorescent secondary antibodies were applied. These included goat anti-rabbit (green dye, Alexa 488, Invitrogen A11034 [GenBank] ), goat anti-mouse (Texas Red, Alexa 594, Invitrogen A21125 [GenBank] ), and goat anti-rabbit (Texas Red, Alexa 555, Invitrogen). Microscopy was performed utilizing standard and confocal microscopes (Olympus, Provis AXZ0TRF and Bio-Rad Radiance 2100 confocal microscopes).

Statistical Analysis

Statistical analysis was performed using SigmaStat (V2.03S, Systat Software, Point Richmond, CA). Physiological, RNA, and protein data were analyzed independently. Data were assessed by a two-way ANOVA for time, group, and interaction effects. When a significant interaction effect was observed, post hoc analysis was performed using a Tukey test. All protein band densities were normalized to the corresponding beta-actin band density and reported as the relative increase to the corresponding control. Comparison of two means was performed using a Student's t-test. Bonferroni correction for multiple comparisons was utilized when indicated. In all cases, data are presented as means ± SD and a value of P < 0.05 was taken to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Experimental Protocol 1: Acute Recovery from Hemodilution

Acute hemodynamic, cooximetry, and ABG measurements. No differences in MAP or baseline cooximetry or ABG measurements were observed between anemic and control rats (Fig. 1). Following hemodilution, the hemoglobin concentration decreased from a baseline of 124 ± 9 to a minimum value of 57 ± 2 g/l with a comparable decrease in blood oxygen content (Table 2 and Fig. 1; P < 0.05). Methemoglobin levels increased slightly following hemodilution (1.6 ± 0.2%) relative to control values (1.1 ± 0.1%, P < 0.05). After reduction of the fraction of inspired oxygen (FIO2) from 0.50 to 0.21 (room air), there was a significant reduction in the PaO2 and oxygen saturation in both groups (Table 2; P < 0.05). However, the PaO2 in both groups remained above 100 mmHg and hemoglobin saturation remained above 97%, without any significant difference between groups. All other ABG measurements did not differ between anemic and control groups (Table 2).


Figure 1
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  		Fig. 1. Acute hemodilution and recovery did not result in any significant differences in mean arterial blood pressure, pH, or PaCO2 when comparing anemic and control rats (left). During recovery of spontaneous ventilation on room air, there was a drop in PaO2 observed in both anemic and control rats (top right). However, the PaO2 never decreased below 100 mmHg in either group. The hemoglobin concentration decreased significantly from baseline and control rats after hemodilution (right middle). This difference in hemoglobin concentration was maintained up to 24 h after hemodilution (bottom right). *P < 0.05 relative to baseline. #P < 0.05 relative to controls.

 

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  		Table 2. Experimental protocol 1: acute hemodilution: arterial blood gas and cooximetry data

 
Experimental Protocol 2: Hemodilution and 6- to 24-h Recovery

Hemoglobin concentration in rats recovering from acute hemodilution. There was no difference between the baseline hemoglobin concentration in anemic (n = 6; 128 ± 14 g/l) and control rats (n = 6; 129 ± 10 g/l). Following hemodilution, hemoglobin concentrations were maximally reduced at 1 h (64 ± 13 g/l) and remained below baseline and control values at 6, 12, and 24 h (78 ± 13, 84 ± 10, and 79 ± 11, respectively, P < 0.05 for each time; Fig. 1). In control rats, hemoglobin concentrations remained similar to baseline at 6, 12, and 24 h (119 ± 9, 145 ± 9, and 145 ± 16, respectively; Fig. 1).

HIF-1{alpha} protein analysis. Western blot analysis of HIF-1{alpha} protein demonstrated a significant increase in HIF-1{alpha} protein levels in hypoxic retina and cerebral cortex (Fig. 2; 2.1 ± 0.1-fold, n = 4, P < 0.001). Immediately following hemodilution, no relative increase in HIF-1{alpha} protein was observed (Fig. 2). After 18-h recovery, a 1.7 ± 0.2-fold increase in HIF-1{alpha} protein was observed in the cerebral cortex of anemic rats, relative to controls (Fig. 2; P < 0.05).


Figure 2
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  		Fig. 2. A: increased expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) protein levels is observed in the retina and cerebral cortex of rats exposed to hypoxia (10% oxygen). B: immediately following hemodilution (recovery, 0 h), no significant difference was observed between cerebral cortical HIF-1{alpha} levels in anemic and control rats (left). However, after 18 h of recovery, a significant increase in cerebral cortical HIF-1{alpha} protein was observed in anemic rats (right). Control and anemic samples are run on the same gel for each time period. *P < 0.05 relative to controls.

 
Quantitative real-time RT-PCR. There were no differences in cerebral cortical GAPDH, eNOS, or HIF-1{alpha} mRNA levels between control and anemic rats at any time (Fig. 3). Cerebral cortical nNOS and iNOS mRNA levels were significantly increased in the anemic rats at 6 h, relative to controls (1.8 ± 0.5- and 5.1 ± 3.6-fold, respectively). Cerebral cortical VEGF and CXCR4 mRNA were maximally increased in anemic rats at 12 h (2.1 ± 0.7- and 3.7 ± 0.8-fold, respectively). Cerebral cortical EPO mRNA levels were significantly elevated in anemic rats at 12 h, relative to controls (2.7 ± 1.4-fold, P < 0.05 for all; Fig. 3).


Figure 3
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  		Fig. 3. Quantitative RT-PCR demonstrated stable levels of cerebral cortical mRNA for GAPDH, HIF-1{alpha}, and endothelial nitric oxide synthase (eNOS) over the duration of the experiment without any significant differences between anemic and control groups. A significant increase in cerebral cortical neuronal (n)NOS and inducible (i)NOS mRNA levels was observed after 6 h of recovery in anemic rats. Cerebral cortical vascular endothelial growth factor (VEGF), erythropoietin (EPO), and chemokine receptor 4 (CXCR4) were observed after 6 and 12 h of recovery from acute hemodilutional anemia. *P < 0.05, relative to controls.

 
nNOS and VEGF protein analysis. The peak increase in cerebral cortical nNOS protein (155 kDa) was observed at 12 h in both anemic and hypoxic rats (2.0 ± 0.2- and 1.7 ± 0.2-fold, respectively, relative to controls, P < 0.001 for both; Fig. 4). Western blot analysis for cerebral cortical VEGF protein demonstrated two bands near the expected molecular weight of 46 kDa. In hypoxic rats (positive control), there was a significant increase in the density of both bands (total band) at 6 h, relative to controls (Fig. 4; 1.69 ± 0.18-fold, P < 0.020). At 12 h, an increase in the second VEGF band was observed in the cerebral cortex of both anemic and hypoxic rats (Fig. 5; 1.5 ± 0.4- and 1.9 ± 0.4-fold, respectively, P < 0.01 for both). There was no significant increase in eNOS protein band density (140 kDa) in anemic cerebral cortex at 0 and 18 h of recovery (0.9 ± 0.2 and 1.1 ± 0.1, respectively), relative to controls (1.0 ± 0.1 and 1.0 ± 0.3, respectively).


Figure 4
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  		Fig. 4. A: increased cerebral cortical nNOS and VEGF protein levels were observed in anemic rats, relative to beta-actin, following 12 h of recovery. B and C: time course of cerebral cortical nNOS expression in anemic and hypoxic rats (positive control) suggests that the peak increase in nNOS protein occurred between 12 and 24 h of recovery. D: increased cerebral cortical expression of VEGF was identified in 2 bands following exposure to hypoxia or acute anemia. E and F: time course of cerebral cortical VEGF expression demonstrated a maximal increase in the total VEGF band after 6 h of hypoxia exposure. Independent measurement of the second VEGF band density demonstrated a significant increase after 12 h of anemia or hypoxia exposure. *P < 0.05, relative to controls.

 

Figure 5
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  		Fig. 5. A: immunofluorescence staining demonstrating the perivascular distribution of HIF-1{alpha} (green) in relationship to the astrocytic marker GFAP (red) in anemic cerebral cortex. B: high-resolution image of an astrocyte demonstrating nuclear HIF-1{alpha} staining (white arrows). C: nNOS-positive neurons (green) are colocalized in the cerebral tissue around cerebral blood vessels which demonstrate perivascular HIF-1{alpha} staining (red). D: evidence of cytoplasmic colocalization of HIF-1{alpha} and nNOS can be seen (white arrow). White bar = 50 µm.

 
Immunohistochemistry and immunofluorescence staining. Immunohistochemical (data not shown) and immunofluorescence staining demonstrated an increase in staining for HIF-1{alpha} in the vicinity of cerebral blood vessels in the cerebral cortex of anemic rats, which colocalizes with astrocytes (Fig. 5, A and B). Costaining with nNOS and HIF-1{alpha} demonstrated nNOS-positive neurons in the vicinity of blood vessels with perivascular HIF-1{alpha} staining (Fig. 5, C and D).

Immunohistochemistry identified a significant increase in the number of nNOS-positive cells within the cerebral cortex and basal ganglia of anemic rats. These cells had dense cytoplasmic staining for nNOS and resembled neurons with significant axonal processes which often projected to cerebral blood vessels (Fig. 6, A and B). Quantitative analysis demonstrated a threefold increase in the number of nNOS-immunopositive cells in the cerebral cortex (60 ± 24 vs. 17 ± 7 cells per coronal section) and basal ganglia (32 ± 10 vs. 10 ± 3 cells per coronal section) of anemic rats (Fig. 6C; n = 4 rats, P < 0.001 for both). Immunofluorescent studies identified that the nNOS-positive cells within the cerebral cortex were approximated to blood vessels staining positive for smooth muscle actin (Fig. 6D). nNOS-positive cerebral cortical cells and basal ganglia costained for NeuN (neurons) but not for the astrocytic marker GFAP (Fig. 7, A and B).


Figure 6
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  		Fig. 6. A: immunostaining for nNOS identified a significant increase in specific nNOS-positive cells within the cerebral cortex and basal ganglia. B: quantitative analysis demonstrated a 3-fold increase in nNOS-positive cells in the cerebral cortex and basal ganglia of anemic rats. *P < 0.001. C: nNOS staining demonstrates that nNOS-positive axons project toward cerebral blood vessels (white arrows). D: nNOS-positive cell (green) is closely approximated to a cerebral blood vessel stained red with smooth muscle cell actin. White bar = 50 µm.

 

Figure 7
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  		Fig. 7. A: double labeling studies demonstrated that the nNOS-positive cells (green) costained with the neuronal marker (NeuN, red) as indicated by the white arrows. B and C: nNOS-positive cells (green) in the cerebral cortex and basal ganglia did not costain with the astrocytic marker GFAP (red). Generalized nuclear staining is identified in blue (ToPro3). White bar = 50 µm.

 
Increased staining for VEGF was observed in perivascular regions of the cerebral cortex, relative to controls (data not shown). Immunofluorescence colabeling demonstrated VEGF staining in the perinuclear region in cells that were independently labeled with the neuronal marker NeuN or the astrocytic marker GFAP (Fig. 8, A –D). In anemic cerebral cortex, VEGF-positive cells were often identified in pairs, in which one cell costained with NeuN while the other was NeuN negative (Fig. 8C). In anemic rats, cerebral cortical cells, which costained with VEGF and GFAP, were more prominent than in controls (Fig. 8, B and D).


Figure 8
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  		Fig. 8. A-D: immunofluorescence staining for VEGF (green) in control and anemic rats demonstrated an increase in VEGF-positive cells in the anemic cerebral cortex. VEGF-positive cells colabeled with either the neuronal marker NeuN (A and C, red) or the astrocytic markers GFAP (B and D, red) (horizonatal white arrows). Corresponding vertical arrows represent VEGF-positive cells, which do not colocalize with NeuN or GFAP. White bar = 50 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Despite evidence of cerebral morbidity associated with acute hemodilution (18, 25, 51), little is understood about the regulatory mechanisms that are activated in the brain during acute anemia. The current study demonstrates an increase in HIF-1{alpha} cerebral cortical protein levels in anemic rats, in the absence of increased HIF-1{alpha} mRNA levels. Increased cerebral cortical mRNA levels for HIF-1-responsive molecules including VEGF, EPO, iNOS, and CXCR4 were also observed, suggesting that the HIF-1{alpha} was biologically active in the cerebral cortex of anemic rats. Cerebral cortical nNOS mRNA and protein levels were also increased in anemic rats. These data suggest that cerebral tissue hypoxia may have occurred in anemic rats. Recent experimental evidence demonstrated that hypoxia leads to activation of a novel nNOS promoter, which may be HIF-1 responsive, producing an mRNA with high translational efficiency (50). Following acute exposure to hypoxia, this promoter is activated in specific tissues including the cerebral cortex. There was an increased number of nNOS-positive neurons identified within the cerebral cortex of anemic rats, which frequently extended axonal projections to cerebral blood vessels. These data suggest that NO from cerebral cortical neurons may play a role in vascular signaling during anemia (16). Increases in cerebral cortical HIF-1{alpha} and VEGF protein expression also occurred in the perivascular region of cerebral blood vessels. HIF-1{alpha} and VEGF independently colabeled with both neurons and astrocytes, identifying both cell types as potential sources of these proteins during anemia. The increase in HIF-1{alpha}, nNOS, and VEGF protein in the cerebral cortex of anemic rats may constitute adaptive physiological responses to acute hemodilutional anemia.

The increase in cerebral cortical HIF-1{alpha} protein in anemic rats occurred without a corresponding change in HIF-1{alpha} mRNA level, suggesting that the increase was regulated at the protein level. This mechanism of regulation is consistent with the upregulation of HIF-1{alpha} by hypoxia. Hypoxic HIF-1{alpha} stabilization and transactivation would lead to the regulation of HIF-1-responsive genes, including VEGF, EPO, iNOS, and CXCR4 (2, 31, 41). In this paradigm, cerebral cells may be acting as sensitive biosensors of cerebral tissue hypoxia, which has been previously difficult to measure using tissue oxygen sensors (20, 43, 47). In addition to tissue hypoxia, nonhypoxic mediators can increase HIF-1{alpha} mRNA transcription and protein translation, resulting in an accumulation of the HIF-1{alpha} protein under normoxic conditions (10, 40). The current study cannot exclude the possibility that nonhypoxic mediators contributed to the increase in HIF-1{alpha} protein in anemic cerebral cortex.

The importance of defining the mechanism of HIF-1{alpha} accumulation is emphasized by the finding that NO may influence HIF-1{alpha} levels differentially under normoxic and hypoxic conditions (3, 10). Under hypoxic conditions, NO has been shown to inhibit HIF-1{alpha} and VEGF accumulation (1, 22, 44). Conversely, under normoxic conditions, NO may lead to HIF-1{alpha} protein accumulation (32, 36, 48). The interactive biology of NO and HIF-1{alpha} may regulate adaptive changes to optimize HIF-1-dependent mechanisms of organ protection, which may promote organism survival under conditions of reduced tissue oxygen delivery (26, 27). Conversely, the HIF-1{alpha} may activate pathophysiological mechanisms, including BNIP3, which may induce neuronal apoptosis.

No increases in eNOS mRNA or protein were observed in our model. This is consistent with findings from an earlier study in which hemodilution did not increase eNOS mRNA levels in the cerebral cortex (20). In another study, hemodilution did not increase in periaortic NO or eNOS protein levels unless a high viscosity colloid was utilized to perform severe hemodilution to a low hematocrit of 11% (46). Thus eNOS does not seem to be the predominant NOS isoform activated in our anemia model.

The current study demonstrated that both neurons and astrocytes exhibit an increase in VEGF expression in the cerebral cortex of anemic rats. This is consistent with a hypoxic response (9, 26, 33), which may play a protective role by promoting neuronal survival and angiogenesis (5, 29). Dynamic changes in capillary density have been demonstrated in response to hypoxia (37) favoring optimal delivery of oxygen to cerebral tissue. Evidence of increased VEGF expression and enhanced angiogenesis has also been demonstrated in response to anemia (13, 38). Therefore, the increase in VEGF expression observed in the cerebral cortex of anemic rats may represent activation of proangiogenic and/or neuroprotective mechanisms directed at optimizing cerebral oxygen delivery and neuronal viability. Conversely, increased VEGF may have deleterious effects, including increased vascular permeability (15, 42).

The morphology of the nNOS-positive cerebral cortical neurons observed in anemic rats closely resembles those identified following hypoxic exposure (7). Hypoxic upregulation of cerebral nNOS expression has been identified in primitive vertebrates as well as mammals suggesting that this response may have been conserved through evolution (7, 17, 39). In rodents, hypoxia leads to increased nNOS expression in neurons of the cerebral cortex (7), cerebellum (17), and basal ganglia (14), demonstrating that it may be physiologically important in many regions of the brain. Potential roles for cerebral nNOS include central regulation of myocardial function, vascular tone (35, 49), ventilation (28), and CBF (23, 52). The proximity of nNOS-positive neurons to blood vessels containing smooth muscle actin supports a role for nNOS in vascular signaling, which may help to maintain the delicate balance between cerebral oxygen supply and neuronal metabolic requirements (45, 52). Conversely, excessive nNOS activity may cause NO-mediated neurotoxicity. Given the evidence of increased nNOS and iNOS expression in anemic cerebral cortex, future studies will be required to determine whether these responses contribute to increased oxidative stress within the brain of anemic animals.

There are some limitations of this study. Our model of hemodilution involved 50% hemodilution with pentastarch. Although this colloid is used clinically, it is not used in these proportions. This method allowed us to achieve target hemoglobin concentrations while maintaining MAP and arterial oxygen tension, without the use of vasopressors. Changes in plasma and blood viscosity were not measured in this study. In a comparable model of 50% hemorrhage and resuscitation with 10% pentastrach, plasma viscosity remained near that of controls (1.1 cP) while hemodiluted whole blood viscosity was reduced (1.8 cP) relative to control whole blood (4.2 cP) (4). It is possible that shear stress associated with hemodilution contributed to the observed changes in protein levels. However, in control rats in which isovolemic blood exchange was performed, no increase in cerebral cortical HIF-1{alpha} or nNOS protein levels was observed, suggesting that shear forces were not solely responsible for the observed effects of hemodilution.

In conclusion, interaction between nNOS, HIF-1{alpha}, and VEGF physiology has been established in other experimental paradigms (11, 19, 48). Our data have demonstrated increased expression of cerebral cortical HIF-1{alpha}, nNOS, and VEGF in response to acute hemodilution. The increase in nNOS was colocalized to neurons while VEGF expression colocalized to neurons and astrocytes. Expression of HIF-1{alpha}, nNOS, and VEGF was identified in the perivascular region of the cerebral cortex. These data will help to define important physiological and/or pathophysiological mechanisms activated within the brain during anemia.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work is supported by the Anemia Institute for Research and Education, Canadian Anesthesiologists' Society, Physicians' Services Incorporated Foundation, Department of Anesthesia, St. Michael's Hospital (Toronto, Canada). Dr. Marsden received support from the Heart and Stroke Foundation of Canada (T-4632). Dr. Hare is the recipient of the Bristol-Myers Squibb-CAS Career Scientist Award.

This work was presented in part at the International Anesthesia Research Society, Tampa, FL, in March 2004 and the Society for Neuroscience, San Diego, CA, in November 2004.


   ACKNOWLEDGMENTS
 
This manuscript is dedicated to Marion Hare. The authors acknowledge the expert technical assistance provided by C. Coackley, R. Qu, M. Cheung, and C. Choong. Appreciation is expressed to C. Kay who assisted with the statistical analysis and J. Trogadis for assistance in bioimaging.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. M. T. Hare, Dept. of Anesthesia and Physiology, Univ. of Toronto, St. Michael's Hospital, 30 Bond St., Toronto, Ontario, M5B 1W8, Canada (e-mail: hareg{at}smh.toronto.on.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 REFERENCES
 

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