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Keywords:

  • apoptosis;
  • inflammation;
  • intermittent hypoxia;
  • platelet-activating factor;
  • sleep-disordered breathing;
  • spatial learning

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

Intermittent hypoxia (IH) during sleep, a hallmark of sleep apnea, is associated with neurobehavioral impairments, regional neurodegeneration and increased oxidative stress and inflammation in rodents. Platelet-activating factor (PAF) is an important mediator of both normal neural plasticity and brain injury. We report that mice deficient in the cell surface receptor for PAF (PAFR–/–), a bioactive mediator of oxidative stress and inflammation, are protected from the spatial reference learning deficits associated with IH. Furthermore, PAFR–/– exhibit attenuated elevations in inflammatory signaling (cyclo-oxygenase-2 and inducible nitric oxide synthase activities), degradation of the ubiquitin–proteasome pathway and apoptosis observed in wild-type littermates (PAFR+/+) exposed to IH. Collectively, these findings indicate that inflammatory signaling and neurobehavioral impairments induced by IH are mediated through PAF receptors.

Abbreviations used
IH

intermittent hypoxia

iNOS

inducible nitric oxide synthase

LPP

lateral perforant pathway

LTP

long-term potentiation

MPP

medial perforant pathway

NOS

nitric oxide synthase

PAF

platelet-activating factor

PAFR

PAF receptor

PBS

phosphate-buffered saline

PGE2

prostaglandin E2

RA

room air

Obstructive sleep apnea, a clinical syndrome characterized by repeated episodes of upper airway obstruction during sleep, is now recognized as a highly prevalent public health problem that frequently imposes substantial cardiovascular and neurocognitive morbidities (Young et al. 2002). Indeed, obstructive sleep apnea patients exhibit substantial memory and executive functional losses, have increased circulating markers of oxidative stress and inflammation and develop regional gray matter loss (Montplaisir et al. 1992; Beebe and Gozal 2002; Carpagnano et al. 2002; Macey et al. 2002). The unique regional brain susceptibility in obstructive sleep apnea led to the hypothesis that the behavioral consequences and neural damage are mediated by cyclical exposures to hypoxia–reoxygenation during sleep. In recently developed rodent models, exposure to intermittent hypoxia (IH) leads to neurodegenerative changes, increased oxidative stress and inflammation and impaired spatial learning in the Morris water maze in the absence of significant sleep fragmentation (Gozal et al. 2001; Row et al. 2002, 2003; Li et al. 2003).

Platelet-activating factor (PAF), a potent pro-inflammatory phospholipid messenger that is synthesized in the CNS, has been implicated in neurodegenerative mechanisms including excitoxicity, free radical production, excessive nitric oxide production and regulation of pro-inflammatory cytokine gene induction and release (Bazan et al. 1994; Maclennan et al. 1996; Bazan 1998a; Bennett et al. 1998; Marrache et al. 2002). For example, PAF accumulates in cerebral hypoxia/ischemia and PAF receptor (PAFR) antagonists are neuroprotective (Panetta et al. 1987; Kumar et al. 1988; Pettigrew et al. 1995; Ohmori et al. 1996). However, in addition to its pathophysiological significance, PAF has been implicated in synaptic plasticity and memory formation (Clark et al. 1992; Izquierdo et al. 1995; Bazan 1998b). Indeed, administration of PAF agonists and antagonists modulates long-term potentiation (LTP) in the hippocampal slice preparation and modifies performance during learning and memory task paradigms (Izquierdo et al. 1995; Teather et al. 1998, 2001).

The cloning of the seven transmembrane G-protein-coupled PAFR and the generation of mice deficient in this receptor (PAFR–/–) provide unique tools to examine the hypothesis that activation of PAFRs participates in IH-induced signaling responses (Honda et al. 1991, 2002; Bito et al. 1992; Izumi et al. 1995). Although PAFR–/– mice have been shown to display attenuated LTP, the performance of these receptor-deficient mice on learning and memory tasks has not been explored (Chen et al. 2001). Therefore, the goals of this study were to examine the potential role of PAFR on two different spatial learning and memory tasks in the water maze and to determine whether absence of PAFR decreases inflammatory responses in the cortex following IH exposure and preserves function.

Animals

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

Experiments were performed on 30 male 12-week-old PAFR–/– and 21 wild-type littermate mice (PAFR+/+ 25–30 g). Animals were housed in standard clear polycarbonate cages with food and water available ab libitum, kept on a 12-h light/dark schedule (06:00–18:00 h) and all testing was conducted during the light phase. Experimental protocols were approved by the Institutional Animal Use and Care Committee and are in close agreement with the NIH guide for the care and use of laboratory animals.

Intermittent hypoxia exposures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

Animals were maintained in either IH (n = 30) or room air (RA; n = 21) in identical chambers (30 × 20 × 20 in; Oxycycler model A44XO; BioSpherix, Redfield, NY, USA) for 14 days prior to testing. Oxygen concentration was continuously measured by an O2 analyzer and was controlled by a computerized system, as described previously (Gozal et al. 2001), such as to generate either a cyclical pattern of 5.7 and 21% oxygen every 90 s (IH) during the sleep cycle or 21% oxygen throughout (RA). Ambient temperature was kept at 22–24°C.

Behavioral testing

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

Morris water maze testing was conducted in a white circular pool maintained at a temperature of 21°C. A Plexiglas escape platform was positioned 1 cm below the water surface. Fixed extramaze cues surrounded the maze and were visible to the mice. Maze performance was recorded by a suspended video camera and interfaced with a video tracking system (HVS Imaging, Hampton, UK). Behavioral parameters were analyzed by anova and Student-Newman-Keuls' post-hoc tests as appropriate.

A standard place-training reference memory task was conducted on mice in the water maze following exposure to 14 days of IH or RA. One day prior to place learning, mice were habituated to the water maze during a free swim. Place learning was then assessed over six consecutive days using a spaced training regimen that has been demonstrated to elicit optimal learning in mice (Gerlai and Clayton 1999). Each place-training session consisted of three trials separated by a 10-min intertrial interval. On a given daily session, each animal was placed into the pool from one of four quasirandom start points (N, S, E or W) and allowed up to 90 s to escape to the platform where it was allowed to stay for 15 s. The platform was removed 24 h after the final training session to obtain measures of spatial bias (probe trial).

Spatial working memory in mice was tested using a modified version of the water maze following completion of the standard place-training task (Hodges 1996). During each daily session, mice received four trials, the first trial (information trial) and three subsequent trials (retention trials). The time between trials 1 and 2 was 120 min and that between each successive trial was 30 s. For each subsequent four-trial session, the hidden platform was relocated to a different quadrant and location. The differences in escape latency and pathlength between trials 1 and 2 were used as a measure of working memory.

Nitric oxide synthase (NOS) activity assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

NOS activity was determined by measuring the conversion of l-arginine to l-citrulline using a commercial assay (Cayman, Ann Arbor, MI, USA). Cortical tissues were homogenized in buffer, protein concentrations determined by the Lowry protein assay (Bio-Rad, Hercules, CA, USA) and 100 µg proteins were incubated in reaction mixture (total volume 50 µL) containing 50 mm TrisHCl (pH 7.4), 1 mm NADPH, 5 mm FAD, 5 mm FMN, 15 mm BH4 and purified [3H]l-arginine (1 µCi) at 30°C for 60 min. To determine calcium-independent NOS (iNOS) activity, the assay was conducted in the presence of 1 mm EGTA without calcium and calmodulin. The reaction was stopped by adding 400 µL of stopping buffer (50 mm HEPES and 5 mm EDTA) followed by the addition of 100 µL resin. Following centrifugation, eluates were transferred to vials for liquid scintillation counting.

Prostaglandin E2 enzyme immunoassay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

Cortical tissue concentrations of prostaglandin E2 (PGE2) were determined using an enzyme immunoassay kit (Oxford Biomedical Research, Oxford, MI, USA). Tissues were homogenized in 15% methanol in 0.1 m sodium phosphate buffer (pH 7.4). After brief centrifugation, the supernatant fluid was diluted with 0.1 m phosphate buffer (pH 7.0) and transferred to activate ODS-silica reverse phase Sep-Pak C18 columns (Waters, Milford, MA, USA). The columns were rinsed with 2 mL of 15% methanol followed by 2 mL petroleum ether and the PGE2 eluted with 2 mL methyl formate. The methyl formate eluate was evaporated and resuspended in 1 mL of extraction buffer. The PGE2 concentration was then determined spectrophotometrically in duplicate after incubation with PGE2 enzyme conjugate and substrate in a microplate.

Proteasomal activity assays

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

Unfixed tissues corresponding to the CA1 region of the hippocampus were homogenized by sonication in ice-cold 50 mm Tris-HCl (pH 7.4) containing 1 mm EDTA. Homogenates corresponding to the two hippocampi in each animal were pooled and immediately used for the fluorometric determination of the 20S proteasomal enzymatic activity as previously described (Craiu et al. 1997) with 75 µm Suc-Leu-Leu-Val-Try-AMC, Boc-Leu-Arg-Arg-AMC and Z-Leu-Leu-Glu-AMC as substrates to measure the chymotrypsin-like, trypsin-like and peptidyl glutamyl-peptide hydrolytic activities, respectively, expressed as fluorescence units/min/µg brain tissue protein. Protein concentrations were determined as above and expressed as mg protein/tissue weight. The specificity of proteasomal assays was established by the ability of 20 µm lactacystin, a selective inhibitor of proteasomal function, to block fluorescence change (Craiu et al. 1997).

Immunohistochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

Four groups of mice (PAFR+/+ and PAFR–/– exposed to either RA or IH for 7 days, n = 4/group) were anesthetized with pentobarbital (50 mg/kg i.p.) and perfused transcardially with 50 mL phosphate-buffered saline (PBS) and then with 2.5% paraformaldehyde in cold PBS containing 5% sucrose, pH 7.4. The brain was removed and placed overnight in a fixative containing 1% paraformaldehyde in PBS and 30% sucrose at 4°C. Coronal sections (40 µm) were washed in PBS, incubated in 0.4% triton X-100 in PBS containing 1.5% normal goat serum for 1 h and incubated with cleaved caspase 3 antibody (1 : 2500; Cell Signaling, Beverly, MA, USA). Once the primary antibody reaction was completed, sections were washed in PBS, incubated in biotinylated anti-rabbit IgG (Vector, Burlingame, CA, USA) diluted in 0.4% triton X-100 in PBS for 1 h, washed three times in PBS, incubated for 1 h in avidin-biotinylated horseradish peroxidase (Vectastain Elite kit; Vector) diluted in 0.4% triton X-100 in PBS, rinsed three times in Tris (pH 7.6) and incubated in 50% diaminobenzidine tetrahydrochloride and 0.005% H202 diluted in Tris (pH 7.6) for 1–3 min. The reaction was stopped in PBS and sections were mounted and examined using a Nikon Ellipse E800 microscope and a SPOT digital camera. Ten adjacent sections were assessed for every animal and the number of cleaved caspase 3 positively labeled cells was tabulated.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

Intermittent hypoxia impairs spatial reference memory in PAFR+/+ but not PAFR–/– mice. On a standard place-training discrimination task, PAFR–/– mice displayed normal spatial learning compared with wild-type littermates (Fig. 1). anova revealed a significant genotype × treatment interaction for both latency [F(3,47) = 5.019, p < 0.005] and pathlength [F(3,47) = 5.019, p < 0.005] and post-hoc analyses revealed that wild-type mice exposed to IH were impaired compared with all other treatment groups on measures of task acquisition (p < 0.05). No significant group differences were observed over the final 2 days of training (block 3, Fig. 1), indicating that all groups were able to reach a similar level of performance by the end of training. Similarly, no significant differences emerged in swimming speeds over the trials among the two groups. On measures of spatial bias obtained during the probe trial conducted 24 h following completion of training, no significant group differences were observed in the percentage of time spent in the platform quadrant or in the relative proximity to target platform location (Fig. 1). anova revealed a significant genotype × treatment interaction on the number of platform crossings [F(3,47) = 2.903, p < 0.045] and post-hoc analyses revealed that wild-type mice exposed to IH had fewer platform crossings than any other group (Fig. 1).

image

Figure 1. Mice deficient in platelet-activating factor receptors (PAFRs) are protected from intermittent hypoxia (IH)-induced spatial learning deficits. (a) Mean latencies (s) and swim distances (cm) to locate the target platform during place training in mice deficient in PAFRs ( PAFR –/–) or wild-type littermates ( PAFR +/+) exposed to 14 days of IH ( PAFR –/– IH, ^ PAFR +/+ IH, □) or room air (RA; PAFR –/– RA, • PAFR +/+ RA, ▪). (b) Probe trial performance of PAFR –/– exposed to 14 days of IH (▥) or RA (bsl00017) and of PAFR +/+ (□, RA; ▪, IH) on measures of spatial bias. Proximity to target platform location (cm), percentage of time spent in the target quadrant and mean number of target platform crossings are shown. Data are expressed as mean ± SE; * p  < 0.01 versus PAFR +/+ RA.

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Working memory spatial impairments are associated with exposure to IH and with absence of PAFR expression. On the working memory task, PAFR–/– mice were impaired compared with their wild-type littermates and IH exposures were associated with working memory impairments in PAFR–/– and PAFR+/+ mice (Fig. 2). anova assessing the savings from trial 1 (the information trial) to trial 2 (the retention trial) revealed significant genotype × treatment interactions for both latency [F(3,47) = 3.100, p < 0.036] and pathlength [F(3,47) = 5.342, p < 0.003]. Post-hoc analyses revealed that normoxic PAFR+/+ mice had greater savings compared with other groups (p < 0.05).

image

Figure 2. Spatial working memory impairments are associated with exposure to intermittent hypoxia (IH) and with absence of platelet-activating factor receptor (PAFR) expression. Mean latency (s) and pathlength (cm) savings from trial 1 to trial 2 on a working memory task in mice deficient in the PAFR ( PAFR –/–) or wild-type littermates ( PAFR +/+) exposed to 14 days of IH ( ▥ and ▪, respectively) or room air (RA; zbsl00017 and □, respectively). Data are expressed as mean ± SE; * p  < 0.01 versus PAFR +/+ RA.

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Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

Intermittent hypoxia induced an elevation in cortical iNOS activity in wild-type mice (vs. RA). However, no differences in either total NOS or iNOS activities were observed in PAFR–/– mice exposed to IH or RA (Fig. 3a). anova revealed significant genotype × treatment interactions [F(3,12) = 3.598, p < 0.046] and post-hoc analyses showed that only wild-type mice exposed to IH demonstrated elevated iNOS activity (p < 0.05).

image

Figure 3. Mice deficient in platelet-activating factor receptors (PAFRs) display attenuated inflammatory responses to intermittent hypoxia (IH). (a)  Mean iNOS activity in mice deficient in the PAFR ( PAFR –/–) or wild-type littermates ( PAFR +/+) exposed to 14 days of IH ( ▥ and ▪, respectively) or room air (RA; bsl00017 and □, respectively). Data are expressed as percent of RA (mean ± SE, n  = 4; * p  < 0.01 vs. PAFR +/+ RA). (b)  Prostaglandin E2 (PGE2) concentrations in cortical tissue of mice deficient in the PAFR ( PAFR –/–) or wild-type littermates ( PAFR +/+) exposed to 14 days of IH ( ▥ and ▪, respectively) or RA ( bsl00017 and □ respectively). Data were expressed as ng/mg protein (mean ± SE, n  = 5; * p  < 0.01 vs. PAFR +/+, # p  < 0.01 vs. PAFR –/–).

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Mice deficient in the PAFR display a marked reduction in IH-induced PGE2 concentrations. PGE2 concentrations were increased after exposure to IH in both PAFR+/+ and PAFR–/– mice [F(3,16) = 23.316, p < 0.001]., but the magnitude of the increase was greater in PAFR+/+ mice compared with PAFR–/– mice (p < 0.01). No significant differences in PGE2 levels were observed between normoxic PAFR+/+ and PAFR–/– (Fig. 3b).

Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

The cortical chymotrypsin-like and trypsin-like proteasomal-like activities of IH PAFR+/+ (p < 0.001) and PAFR–/– mice (p < 0.05) were reduced compared with normoxic conditions (Fig. 4; n = 6/group). However, peptidyl glutamyl-peptide-like activity was reduced by IH only in wild-type mice. Furthermore, the declines in proteasomal activity with IH were significantly greater in PAFR+/+ mice compared with PAFR–/– mice (p < 0.01) in the absence of differences in tissue protein content (Fig. 4).

image

Figure 4. Mice deficient in the platelet-activating factor receptor (PAFR) and exposed to intermittent hypoxia (IH) have preserved proteasomal activity . Chemotrypsin-like (CT), trypsin-like (TR) and peptidyl glutamyl-peptide-like (PDP) proteasomal activities and protein content of cortical tissues of mice deficient in the PAFR ( PAFR –/–) or wild-type littermates ( PAFR +/+) exposed to 14 days of IH (▥ and ▪, respectively) or room air (RA; bsl00017 and □, respectively). * p  < 0.01 vs. PAFR +/+ RA, # p  < 0.05 vs. PAFR –/– RA; PAFR +/+ vs. PAFR –/– p  < 0.01 anova . No differences in protein content were present among the four treatment groups. ( n  = 6/treatment group). FU, fluorescence unit.

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Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

Both PAFR+/+ and PAFR–/– mice exhibited substantial increases in the overall number of cells expressing cleaved caspase 3 within the prefrontal cortex and CA1 region of the hippocampus following 7 days IH (Fig. 5). However, such increases were significantly attenuated in PAFR–/– mice (p < 0.01; Fig. 5).

image

Figure 5. Mice lacking platelet-activating factor receptor (PAFR) have reduced expression of cleaved caspase 3 in prefrontal cortex following intermittent hypoxia (IH) exposure. (a) Cortical section from prefrontal cortex immunostained for cleaved caspase 3 expression after exposure to IH for 7 days, showing positively labeled cells (e.g. arrow). Scale bar, 20 µm. (b) Number of cleaved caspase 3 positively stained cells in prefrontal cortex after 7-day exposure of PAFR +/+ or PAFR –/– mice to either room air (RA) or IH ( n  = 4/group). Data are expressed as mean number of positively stained cells of caspase 3/10 sections. Cleaved caspase 3 positively labeled cells were significantly increased in IH ( p <  0.001 vs. RA). However, PAFR –/– mice exposed to IH exhibited reduced populations of cells expressing cleaved caspase 3 compared with PAFR +/+ (* p  < 0.01). WT, wild type; KO, knockout.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References

This study shows that IH-induced deficits in the acquisition of a spatial task are mediated, at least in part, by activation of PAFR, downstream induction of COX-2 and iNOS activities and decreased proteasome function, ultimately leading to neuronal apoptosis. Furthermore, PAFRs appear to be critical to spatial working memory tasks in the mouse but do not appear to modify the acquisition of a standard spatial reference task in the water maze.

Intermittent hypoxia during sleep imposes neurobehavioral consequences that stem, at least in part, from activation of inflammatory signaling cascades. These cascades have been mechanistically implicated in the neurodegeneration of traumatic brain injury or Alzheimer's disease (Nogawa et al. 1997; Andreasson et al. 2001). Hypoxia/ischemia promotes the accumulation of free arachidonic acid in brain through phospholipase A2 activation (Bazan 1970). More recently, we have shown that increased expression and activity of COX-2 and 15-isoprostane F2 (Row et al. 2003) occur during IH exposures and induce neurobehavioral deficits (Li et al. 2003). Such increases in COX-2 occurred in PAFR+/+ mice following IH. COX-2 induction and subsequent synthesis of PGE2 and other eicosanoids from arachadonic acid suggest that COX-2 is an important mediator of injury, although the mechanism of action remains undefined at present. Platelet-activating factor, a potent proinflammatory messenger induced by phospholipase A2 activation, is elevated in response to hypoxia/ischemia and has been proposed to contribute to cellular injury via exacerbation of glutamate neurotoxicity as well as augmenting the expression of COX-2 (Bazan et al. 1994; Pettigrew et al. 1995; Bazan 1998a). Hypoxia leads to excessive release of glutamate and activation of glutamatergic receptors and PAF antagonists attenuate glutamate excitoxicity as well as afford neuroprotection in models of cerebral hypoxia and ischemia (Rothman 1984; Panetta et al. 1987; Ohmori et al. 1996). Both PAF neurotoxicity and its modulation of synaptic plasticity appear to be mediated by a synaptic and an intracellular form of PAFR; however, only the cell surface receptor has been cloned to date (Honda et al. 1991; Bazan 1998a). Mice with a targeted deletion of the cloned PAF cell surface plasma membrane receptor gene do not display any overt abnormalities in their development, somatic growth or reproduction (Ishii et al. 1998). In the present study, we found that these mice were protected from IH-induced learning deficits on a spatial reference memory task. This protection was associated with attenuated NOS activity and PGE2 responses to IH, smaller reductions in proteasome function and reduced numbers of apoptotic cells, providing further support for the role of PAF in pathophysiological signaling processes induced by IH. While the exact role of PAF release and activation of PAFR during IH remains unclear, targeted ablation of the receptor unequivocally demonstrates that it modulates the downstream activity of COX-2 and iNOS and that, either directly or via these inflammatory mediators, PAFR mediates components of the IH-induced neuronal injury.

Normoxic PAFR–/– mice display normal learning on a spatial reference task but are impaired on a working memory version of the task, indicating that PAFRs play a heterogeneous role in learning and memory processes as PAF and PAF antagonists have been implicated in memory and behavioral tasks (Izquierdo et al. 1995; Teather et al. 2001). Pre-synaptic, but not the intracellular form of, PAFR antagonists block tetanic stimulation-induced LTP in the Schaffer collateral CA1 neurons, a major excitatory hippocampal synapse (Arai and Lynch 1992; Clark et al. 1992; Kato et al. 1994). The perforant path, the major excitatory input to the hippocampus, is subdivided into the lateral (LPP) and medial (MPP) perforant pathways which originate from separate regions of the entorhinal cortex, namely the lateral and medial entorhinal cortex, respectively. The MPP and LPP innervate the hippocampus in a topographical pattern, suggestive of distinct MPP and LPP inputs (Hjorth-Simonsen and Jeune 1972). Electrophysiological and behavioral studies also indicate that MPP and LPP play distinct roles in learning and memory. Lesions of the MPP, but not the LPP, impair place learning in the water maze (Ferbinteanu et al. 1999). Additionally, mice lacking protein phosphatase 1 display normal place learning despite attenuated LTP within the LPP (Allen et al. 2000). PAFR–/– mice display attenuated LTP in the LPP but normal LTP in the MPP (Chen et al. 2001). Thus, our findings provide further support to the notion that the MPP does not play an important role in spatial learning in the water maze. In contrast, lesions of the LPP are associated with enhanced context fear conditioning and decreased habituation to a novel environment. As would be predicted by the potentially different roles of the hippocampal regions receiving projections from the LPP and the MPP, administration of mc-PAF has indeed been shown to enhance habituation to a novel environment, an LPP-dependent function (Myhrer 1988; Izquierdo et al. 1995). As frontal cortical projections to the lateral entorhinal cortex are heavier than to the medial entorhinal cortex and prefrontal cortical systems appear to be involved in the learning of general task ‘rules’ and behavioral flexibility as well as novelty responses, we would predict that disruption of the LPP–lateral entorhinal cortex pathway would adversely impact working memory while leaving reference memory intact (Eichenbaum et al. 1990; Otto and Eichenbaum 1992; Burwell 2000).

Some technical issues deserve comment, i.e. PAFR mice have a targeted deletion of the cell surface PAFR but not of the intracellular form PAFR (Ishii et al. 1998). Therefore, discrepancies between pharmacological studies (Kato 1999; Kobayashi et al. 1999; Honda et al. 2002) and ability to induce LTP in PAFR–/– mice would suggest that additional PAFRs may be expressed and/or that PAF may operate via alternative pathways that do not require receptor activation. Platelet-activating factor could also modulate learning and memory at locations other than the synaptosomal receptors by altering gene expression through its activity on intracellular forms of the receptor. Of particular note, the IH paradigm employed in this study is of insufficient magnitude to induce necrotic cell death and induces a constellation of mild adverse effects that alone would be highly unlikely to be toxic. However, the cumulative effect of these neural responses to IH clearly overcomes innate defense mechanisms such that blockade of one of the multiple deleterious pathways is sufficient to attenuate the pathological effects of IH (Li et al. 2003; Row et al. 2003). This assumption is further corroborated by the smaller reductions in proteasomal activity in PAFR–/– mice. Impairments in proteasomal activity will lead to inhibition of peptide and protein hydrolysis and, therefore, are associated with accumulation of ubiquitinated proteins as protein aggregates and neuronal cytotoxicity (Canu et al. 2000). Furthermore, the alpha subunits of the 26/20S proteasomes are particularly vulnerable to oxidative stress (Bulteau et al. 2001) and IH is clearly associated with marked increases in reactive oxygen species formation (Row et al. 2003). Thus, it is likely that reductions of apoptosis in PAFR–/– mice exposed to IH reflect the improved preservation of proteasome function and reduced oxidative stress.

In conclusion, IH-induced inflammatory signaling, apoptosis and spatial learning deficits are mediated, at least in part, via activation of cell surface pre-synaptic PAFRs. Furthermore, PAFRs appear to play a predominant role in working memory.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Generation and genotyping of deficient mice and wild-type littermates
  5. Animals
  6. Intermittent hypoxia exposures
  7. Behavioral testing
  8. Nitric oxide synthase (NOS) activity assay
  9. Prostaglandin E2 enzyme immunoassay
  10. Proteasomal activity assays
  11. Immunohistochemistry
  12. Results
  13. Intermittent hypoxia-induced increases in inducible nitric oxide synthase (iNOS) activity are absent in platelet-activating factor receptor-deficient mice
  14. Mice deficient in the platelet-activating factor receptor and exposed to intermittent hypoxia have preserved proteasomal activity
  15. Mice lacking platelet-activating factor receptor have reduced expression of cleaved caspase 3 in prefrontal cortex
  16. Discussion
  17. Acknowledgements
  18. References
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