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Keywords:

  • CD44;
  • cerebral ischemia;
  • interleukin-1β;
  • inflammation;
  • mouse;
  • neuroprotection

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

CD44 is a transmembrane glycoprotein known to be involved in endothelial cell recognition, lymphocyte trafficking, and regulation of cytokine gene expression in inflammatory diseases. In the present study, we demonstrated that the expression of CD44 mRNA was induced in a mouse model of cerebral ischemia. A potential role of CD44 in ischemic brain injury was investigated using CD44-deficient (CD44–/–) mice. Over 50% (< 0.05, n = 14) and 78% (< 0.05, n = 10) reduction in ischemic infarct was observed in CD44–/– mice compared with that of wild-type mice following transient (30 min ischemia) and permanent (24 h) occlusion of the middle cerebral artery (MCAO), respectively. Similarly, significant improvement was observed in neurological function in CD44–/– mice as evidenced by spontaneous and forced motor task scores. The marked protection from ischemic brain injury in CD44–/– mice was associated with normal physiological parameters, cytokine gene expression, astrocyte and microglia activation as compared with wild-type mice. However, in CD44–/– mice, significantly lower expression of soluble interleukin-1β protein was noted after brain ischemia. Our data provide new evidence on the potential role of CD44 in brain tissue in response to ischemia and may suggest that this effect might be associated with selective reduction in inflammatory cytokines such as interleukin-1β.

Abbreviations used
CBF

cerebral blood flow

ECA

external common carotid

GFAP

glial fibrillary acidic protein

HA

hyaluronan

ICA

internal common carotid

IL

interleukin

MCAO

occlusion of the middle cerebral artery

TNF

tumor necrosis factor

Cerebral ischemia is a pathophysiological condition induced by the occlusion of cerebral vessels by a thrombus. The resultant deprivation of blood flow, and hence oxygen and glucose, in the ischemic brain area eventually leads to cell death (necrosis and apoptosis), inflammation, and tissue repair and remodeling (del Zoppo et al. 2000; Wang and Feuerstein 2000).

The inflammatory response to brain ischemia has been studied extensively. The early accumulation of neutrophils in ischemic brain has been demonstrated by histopathological (Garcia and Kamijyo 1974; Hallenbeck et al. 1986), biochemical (Barone et al. 1991), and 111In-labeled leukocyte studies (Dutka et al. 1989). Accumulating evidence suggests that the acute inflammatory response after ischemic brain injury is mediated by a sequence of gene expression (Wang and Feuerstein 2000), including inflammatory cytokines, chemokines, cellular adhesion molecules, and other proinflammatory genes (del Zoppo et al. 2000).

CD44 is a transmembrane adhesion receptor and the major cell surface receptor for the non-sulfated glycosaminoglycan hyaluronan (HA) (Aruffo et al. 1990). CD44 plays an important role in lymphocyte adhesion to inflamed endothelium and in tumor metastasis (Lesley et al. 1993). Multiple roles for CD44 in inflammation have been proposed (for review see Puré and Cuff 2001), including leukocyte recruitment, cell–matrix interactions, regulation of leukocyte and parenchymal cell function, metabolism of HA and matrix remodeling. CD44 gene expression was found to be regulated by HA fragments and interleukin-1β (IL-1β) (Foster et al. 2000) and CD44 ligand binding is activated by proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) (Maiti et al. 1998). A critical role for CD44 has been suggested in a number of inflammatory diseases in humans, including rheumatoid arthritis (Mikecz et al. 1999), chronic inflammatory bowel disease (Wittig et al. 1999), atherosclerosis (Cuff et al. 2001) and lung inflammation/fibrosis (Teder et al. 2002). In our previous study, we reported the identification of CD44 up-regulation in response to ischemic brain injury in rat using a subtractive gene hybridization strategy (Wang et al. 2001). CD44 was induced in parallel with the accumulation of leukocytes/macrophages in the ischemic brain tissue, and its expression was localized in microglia/macrophages and brain capillaries in the ischemic lesions (Wang et al. 2001), suggesting a role of CD44 in inflammatory responses following focal stroke as well.

In the present study, we have employed mouse models of focal cerebral ischemia by means of transient and permanent occlusion of the middle cerebral artery (MCAO) in CD44 null mutant mice (CD44–/–) to extend our investigation on the potential significance of CD44 up-regulation. CD44–/– and wild-type (C57BL/6) mice were subjected to MCAO and followed by the analysis of infarct size and neurological deficits. In addition, since CD44 was implicated in cytokine regulation, and cytokines were shown to be expressed de novo in ischemic brain tissue, we have evaluated the expression of two key pro-inflammatory cytokines (IL-1β and TNFα) in CD44–/– and wild-type mice after MCAO.

Focal brain ischemia

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

CD44–/– mice were back-crossed to C57BL/6 over six generations as described previously (Schmits et al. 1997) and produced at the Charles River Laboratories (Wilmington, MA, USA). Adult CD44–/– and wild-type (C57BL/6) mice (18–22 g, paired based on gender and weight) were used for the present study. Animals were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals [DHEW (DHHS) Publication No. (NIH) 85–23, revised 1996, Office of Science and Health Reports, DRR/NIH, Bethesda, MD, USA]. Procedures using laboratory animals were approved by the Institutional Animal Care and Use Committee of Bristol-Myers Squibb Company.

Mice were anesthetized with gas inhalation comprised of a mixture of 30% oxygen (0.3 L/min) and 70% nitrous oxide (0.7 L/min). The gas was passed through an isoflurane vaporizer set to deliver 3–4% isoflurane during initial induction and 1.5–2% during surgery. After shaving the neck and swabbing the surgical site with betadine, an incision of the skin was made directly on top of the right common carotid artery region. The fascia was then blunt dissected until the bifurcation of the external common carotid (ECA) and internal common carotid (ICA) was isolated. A small incision was made on the ECA, and a 5–0 mono-filament suture (9–11 mm long with a round tip) was threaded into the ICA via the ECA. The suture was advanced towards the middle cerebral artery (MCA) to create focal ischemia. In experiments where permanent brain ischemia was studied, the suture was kept in place, while the suture was removed 30 min after MCAO in the transient brain ischemia model. Sham-operation was performed using the same procedure except that no suture was inserted into the carotid artery. Mice were anesthetized with gas inhalation and forebrains were removed at various times following ischemia, reperfusion or sham surgery as indicated in each figure legend. For ELISA analysis, the entire ipsilateral and contralateral hemispheres were dissected and immediately frozen in liquid nitrogen and stored at −80°C for later use.

To measure the infarct volume, brains were removed at 24 h after MCAO and evaluated using 2,3,5-triphenyltetrazolium chloride staining of 2-mm thick brain slices. The stained brain tissue was fixed in 10% formalin in phosphate-buffered saline. The image was captured using a Microtek ScanMaker 4 DUO Scanner (MicroWarehouse Lakewood, NJ, USA) and quantitated using Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD, USA).

Neurological deficits and rota-rod test

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

Neurological deficits were examined at day 1 and 3 after MCAO (n = 10) using a five-point scale as described previously (Wang et al. 2002). Specifically, no neurological deficit = 0; right Horner's syndrome counts as 1 point; failure to extend left forelimb and hindlimb, 1 point each; turning to left, 1 point; and circling to left, 1 point.

The same groups of animals were also subjected to rota-rod test using an Accelerating Speed Treadmill (Stoelting, Wood Dale, IL, USA). Each time, a mouse was given four trials and the mean values were collected for group data analysis.

Physiological parameters

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

The physiological parameters were measured and confirmed under two anesthesia conditions, i.e. gas inhalation as described above and pentobarbital (50 mg/kg, intraperitoneally). In randomly selected animals, regional cerebral blood flow (CBF) was measured with a Laser Doppler Perfusion Monitor (Moor Instruments Inc., Wilmington, DE, USA). After anesthesia, a small incision was made at the midpoint between the right orbit and the external auditory canal. The temporalis muscle was retracted and the underlying fascia cleared. The Laser Doppler probe was placed 1.5 mm posterior and 3.5 mm lateral to the Bregma on the ipsilateral hemisphere. CBF was carefully monitored (to avoid any large vessel) before, during (15 min) MCAO and 30 min after reperfusion.

The arterial blood pressure and heart rate were measured by connecting a tubing through femoral artery using an MP100 Workstation and analyzed using an AcqKnowledge software (BIOPAC Systems, Inc, Santa Barbara, CA, USA) according to the manufacturer's specification. Femoral arterial blood samples were analyzed for pH, oxygen (pO2) and carbon dioxide (pCO2) by direct collection through a PE-50 tubing into an i-STAT G3 + cartridge and processed with a portable clinical analyzer (Abbott Laboratories, Abbott Park, IL, USA).

Real-time RT-PCR

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

Total RNA was isolated from ipsilateral and contralateral brain tissues (n > 4, see figure legends for detail) after transient MCAO or after sham-operation using an RNA isolation kit from Qiagen (Valencia, CA, USA). The primers and probes (Table 1) used for real-time RT-PCR were designed using a Primer-Express 1.0 software from PE Applied Biosystems (Foster City, CA, USA). The specificity of PCR primers for CD44, IL-1β, TNFα and a house keeping gene, rpL32, was tested using a standard PCR protocol in Perkin-Elmer thermocycler of Model 9600 (Foster City, CA, USA) prior to TaqMan quantitation and confirmed by gel electrophoresis. Real-time PCR was performed as described in detail previously (Wang et al. 2001) with the following modifications: One-step RT-PCR was performed using a Gibco BRL PLATINUM Taq System (Gibco BRL, Grand Island, NY, USA) according to the manufacturer's specification. The reaction started with 0.5–1 µg of total RNA at 25 µL reaction volume. The reaction mixture contained 12.5 µL of 2 × Reaction Mix, 0.6 µL of 50 mm MgSO4, 0.125 µL RNase inhibitor, 0.5 µL each of the 10 µm sense and antisense primer, 0.5 µL of the 5 µm probe, and 0.3 µL of RT/Taq Mix. The mixture was incubated in 50°C for 30 min, 95°C for 5 min and then started the PCR cycles at 95°C 15 s and 60°C 60 s for 40 cycles. Each RT-PCR was done in duplicate and performed simultaneously. Data were analyzed using the Sequence Detector V1.6.3 program (Perkin-Elmer).

Table 1.  Primers and TaqMan probes used in the real-time PCR
Primer/probeSequencesPosition (bp)
  1. TNF, tumor necrosis factor; IL, interleukin. The forward (F) and reverse (R) primers as well as probes were synthesized according to the mouse CD44 (GenBank accession No. AJ251594), TNF-α (GenBank accession No. M13049), IL-1β (GenBank accession No. M15131) and rpL32 (GenBank accession No. AK002353) cDNA sequences, respectively. TaqMan probes contains 6-FAM for CD44, IL-1β, TNFα at 5′-end and VIC for the rpL32. All the probes have a quencher dye, 6-carboxytetramethylrhodamine (TAMRA), at the 3′end.

CD44-F5′-CAGCCTACTGGAGATCAGGATGASense 725–747
CD44-R5′-GGAGTCCTTGGATGAGTCTCGAAntisense 2050–29
CD44-probe5′-CTTCTTTATCCGGAGCACCTTGGCCACSense 751–777
TNFα-F5′-TCATGCACCACCATCAAGGASense 1081–1100a
TNFα-R5′-GAGGCAACCTGACCACTCTCCAntisense 1181–1161
TNFα-probe5′-AATGGGCTTTCCGAATTCACTGGAGCSense 1105–1130b
IL-1β-F5′-ACACTCCTTAGTCCTCGGCCASense 976–996
IL-1 β-R5′-CCATCAGAGGCAAGGAGGAAAntisense 1076–1057
IL-1 β-probe5′-CAGGTCGCTCAGGGTCACAAGAAACCSense 1000–1025
rpL32-F5′-TGTCCTCTAAGAACCGAAAAGCSense 360–381
rpL32-R5′-CGTTGGGATTGGTGACTCTGAAntisense 431–411
rpL32-probe5′-TTGTAGAAAGAGCAGCACAGCTGGCCSense 384–409

Enzyme-linked immunosorbent assay for IL-1β

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

To prepare the tissue lysate, ipsilateral and contralateral brain samples (24 h after transient MCAO, n > 7) were pulverized using a porcelain mortar and pestle under liquid nitrogen. The pulverized brain tissues were incubated in a lysis buffer (10 mm Tris pH 8.0, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100 and 5 µL/mL of protease inhibitor cocktail (Sigma, P-8340) for 1 h at 4°C. After 10 min centrifugation at 10 000 × g, the supernatant of tissue lysate was collected and aliquoted for ELISA and protein concentration measurement using a Bio-Rad DC Protein Assay kit (Hercules, CA, USA). The levels of soluble IL-1β protein in the brain tissue were measured using an ELISA kit for mouse IL-1β (Endogen) following the manufacturer's specification. Tissue extracts (50 µL) were applied to each well for the ELISA and the final measure was read out using a plate reader at 450 nm. The concentration of IL-1β in each sample was determined according to the standard (recombinant protein) provided with the kit. Data were at linear part of the standard curve. Each sample was normalized by its protein concentration in milligrams.

Immunohistochemical analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

CD44–/– and wild-type mice were subjected to transient MCAO as described above and the brain tissues were collected at 6, 12, 24 and 48 h post reperfusion (n = 4) for immunohistochemical analysis as described in detail previously (Wang et al. 2001). Primary antibodies used for the present study include rat anti-mouse CD44 (IM7) 1 : 100 dilution; BD PharMingen, San Diego, CA, USA, rat anti-mouse CD11b (IgG) for microglia staining (1 : 200; Serotec Ltd, Oxford, UK) and rat antiglial fibrillary acidic protein (GFAP) for astrocytes (1 : 1000; Chemicon, Temecula, CA, USA). Normal rat IgG (BD PharMingen) was used as negative control. Biotinylated anti-rat IgG (1 : 200; Vector Laboratories) was used as the secondary antibody to incubate with fluorescein streptavidin as described previously (Wang et al. 2001).

Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

The expression of CD44 mRNA following transient brain ischemia was examined using real-time quantitative RT-PCR. Significant up-regulation of CD44 mRNA was observed 12 h after MCAO (3.4-fold increase over sham-operation, p < 0.01, n = 4), reached highest levels at 24 and 48 h (6.2- and 6.4-fold increases, respectively, p < 0.001), and sustained up to 72 h after MCAO (4.5-fold increase, p < 0.01) (Fig. 1).

image

Figure 1. Temporal expression of CD44 mRNA in the brain after MCA occlusion and reperfusion. Mouse brains (n = 4) following sham operation (24 h) or 1, 3, 6, 12, 24, 48 and 72 h after ischemia/reperfusion were used for RNA extraction. Time-dependent expression of CD44 mRNA was examined using TaqMan real-time RT-PCR. Data are illustrated after normalizing with the signal of the house-keeping gene, rpL32. **p < 0.01, ***p < 0.001, compared with sham operated samples.

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CD44 deficiency protects brain from ischemic brain injury

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

Significant reduction in infarct size was observed following transient MCAO in CD44–/– mice compared with the paired wild-type mice (50% reduction, n = 14, p < 0.05; Figs 2a and b). Similar distribution of infarct areas (between striatal and cortical regions) was observed in CD44–/– (69.5% and 30.5% for striatal and cortical areas, respectively, n = 14) and wild-type (71.4% and 28.6%, respectively, n = 14) mice following transient MCAO. The resistance of CD44–/– mice to ischemic brain injury was also supported by significantly superior neurological function in spontaneous and forced motor performance (rota-rod test) (Figs 2c and d). Neurological deficits were significantly reduced in CD44–/– mice 1 and 3 days after transient MCAO, with 30% (p < 0.05) and 33% (p < 0.01) reduction, respectively, compared with wild-type mice (n > 11). Similarly, the rota-rod score was significantly improved in CD44–/– mice after transient MCAO. It also should be pointed out that the accelerating rota-rod test may also reflect in part the improvement in learning and adaptation since all the groups, including sham-operated animals, showed the improvement in the test score (Fig. 2d). Since no difference was observed following sham-operation between CD44–/– and wild-type mice, only wild-type animal data are illustrated to represent the sham-operated group (Fig. 2).

image

Figure 2. CD44 deficiency reduced infarct size and improved motor function following transient cerebral ischemia. Total ischemic lesion (a) and profile of ischemic areas on sequential forebrain slices (2-mm thick) (b) are illustrated as means ± SE in CD44–/– (n = 14) and paired wild-type (C57BL/6; WT; n = 14) mice following transient MCAO. Neurological deficits (c) and rota-rod scores (d) were collected from CD44–/– (n = 11) and wild-type (n = 12) mice at 1 and 3 days after MCAO/reperfusion, or after sham-operation in wild-type mice (n = 11). Because sham-operation produced similar effects on neurological function, only wild-type animal data are illustrated in (c) and (d). *< 0.05, **< 0.01, compared with the wild-type group after MCAO.

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Significant neuroprotection was also observed in CD44–/– mice after permanent MCAO, with 78% reduction in infarct size (n = 10, p < 0.01; Figs 3a and b) and 54% in neurological deficits (p < 0.01; Fig. 3c) in the same group of animals compared with wild-type mice (n = 10) at 24 h after permanent MCAO (Fig. 3).

image

Figure 3. CD44 deficiency reduced ischemic injury following permanent MCAO. Data are illustrated as in Fig. 2 except that permanent MCAO was applied in CD44–/– (n = 10) and wild-type (n = 10) mice, and neurological deficits were collected from the same groups of animals prior to 2,3,5-triphenyltetrazolium chloride-staining. (a) Profile of ischemic areas on sequential forebrain slices; (b) total ischemic lesion; (c) neurological deficits at one day after MCAO. **< 0.01, compared with the wild-type mice.

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Effect on physiological parameters in CD44–/– mice after cerebral ischemia

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

Various physiological parameters including cerebral blood flow, heart rate, arterial blood pressure, pH, blood oxygen (pO2) and carbon dioxide (pCO2) were measured in CD44–/– and wild-type mice before and after transient MCAO (Table 2). No significant difference was observed in CBF, heart rate, and blood gases between CD44–/– and wild-type mice prior to and after MCAO. The only significant difference was the 11% increase in the mean arterial blood pressure in CD44–/– mice compared with wild-type mice 30 min after reperfusion (p < 0.05; Table 2). However, we wish to point out that none of these variables was beyond normal ranges and therefore likely to be of no physiological consequences. In addition, no obvious difference was observed in cardiovasculature and in particular the cerebral vasculature between CD44–/– and wild-type mice as assessed by anatomical and histological (hematoxolin and eosin staining) analysis (data not shown).

Table 2.  Physiological conditions in wild-type and CD44 knockout mice after MCAO
Treatment MCAOCerebral blood flowaHeart rate (min−1)Mean artery blood pressure (mmHg)pCO2 (mmHg)pO2 (mmHg)pHb
  • a

    Illustrated as the arbitrary flux reading. Mice were subjected to 30 min MCAO followed by reperfusion. The physiological data were measured ‘before’ (prior to MCAO), ‘during’ (15 min after MCAO), or ‘after’ (30 min of reperfusion) MCAO. Blood gases shown here were from pentobarbital as anesthesia and all others were under gas inhalation as described in Materials and methods.

  • *

    < 0.05, compared with the wild-type mice.

  • b

    b Decimals rounded to tenth.

Wild typen = 7n = 6n = 6n = 5n = 5n = 5
 Before313 ± 22299 ± 4184 ± 432 ± 2114 ± 127.3 ± 0.0
 During 62 ± 13326 ± 1882 ± 443 ± 3 93 ± 157.3 ± 0.0
 After162 ± 22348 ± 4587 ± 437 ± 1121 ± 67.3 ± 0.0
CD44–/–n = 7n = 6n = 6n = 5n = 5n = 5
 Before333 ± 31376 ± 2582 ± 835 ± 2 95 ± 117.3 ± 0.0
 During 80 ± 8373 ± 2689 ± 448 ± 3113 ± 157.2 ± 0.1
 After173 ± 32434 ± 1897 ± 3*42 ± 3116 ± 117.3 ± 0.0

Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

Figure 4 depicts the mRNA expression of two key inflammatory cytokines, IL-1β and TNFα, in CD44–/– and wild-type mice 12 h after transient MCAO. Significant induction of both cytokine mRNAs was observed in the ipsilateral (ischemic) brain tissues over the contralateral brain tissues in CD44–/– (with 3.3- and 4.6-fold increase for TNFα and IL-1β mRNA, respectively) and wild-type mice (with 3.3- and 3.4-fold increase for the two mRNAs, respectively). However, no obvious difference was noted between CD44–/– and wild-type mice. In contrast, ELISA analysis revealed that the levels of soluble IL-1β protein in the ischemic brain tissues were significantly lower in the CD44–/– mice (1.6-fold increase over the contralateral) than in wild-type mice (3.4-fold increase over contralateral, p < 0.05, or 53% decrease in CD44–/– mice compared with wild-type mice, p < 0.05) 24 h after transient MCAO (Fig. 5). Unfortunately, due to lack of proper reagents, the levels of soluble TNFα protein could not be determined in the present study.

image

Figure 4. Real-time PCR analysis of (a) TNFα and (b) IL-1β mRNA expression in the brain after transient MCAO. Mice were subjected to 30 min MCAO followed by reperfusion. Brain tissues were collected at 12 h after MCAO and used for real-time PCR analysis as described in Fig. 1 legend. Data are illustrated as relative mRNA levels, with a sum of 100% for TNFα or IL-1β, after normalizing with rpL32.

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image

Figure 5. ELISA analysis of soluble IL-1β expression in the brain after transient MCAO. Mice were subjected to 30 min MCAO followed by reperfusion. Brain tissues were collected at 24 h after MCAO and processed for ‘sandwich’ ELISA analysis as described in Materials and methods. The levels of soluble IL-1β protein in the brain (pg/mL) were determined according to the standard using the recombinant IL-1β. Data are illustrated as means ± SE after normalizing with total protein concentration applied to the assay. *< 0.05 compared with the ipsilateral samples of the wild-type animals.

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Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

Immunohistochemical study using brain tissues (n = 4) at 6, 12, 24 and 48 h after transient MCAO demonstrated that microglia were activated in a similar fashion (including temporal and spatial distribution, as well as the morphology) in CD44–/– and wild-type mice (Fig. 6). A similar activation profile was observed for astrocytes using GFAP immunostaining between the knockout and wild-type mice (Fig. 6).

image

Figure 6. Representative immunohistochemical analysis of CD44, CD11b and GFAP expression in the brain 24 h after transient MCAO. CD44–/– (n = 4) and wild-type (n = 4) mice were subjected to 30 min MCAO followed by reperfusion. Brain tissues were collected at 24 h after MCAO and processed for immunohistochemical analysis as described in Materials and methods. Rat anti-mouse CD44 (IM7) was used to detect CD44 expression in wild-type (WT) mice but not in CD44–/– mice (a and b). Anti-CD11b was used to detect microglia (c and d) and anti-GFAP for astrocytes (e and f) in the ischemic lesions.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References

Previously we reported the discovery of CD44 up-regulation in ischemic brain tissue by means of a gene subtraction strategy (Wang et al. 2001). In the present study, we provide novel evidence for the potential role of CD44 in ischemic brain injury using CD44–/– mice subjected to transient and permanent MCAO. While there is no significant alteration in various physiological parameters (including CBF, blood gases and heart rate) observed in CD44–/– mice prior to and after ischemic injury, both infarct size and neurological deficits were markedly reduced in CD44–/– mice compared with the wild-type animals after MCAO. The degrees of different ischemic lesion reduction following transient (50%) and permanent (78%) MCAO in CD44–/– mice might reflect the relatively mild ischemic damage (i.e. 30 min transient occlusion followed by reperfusion) versus a more severe damage (i.e. permanent MCAO).

As CD44 is known to play an important role in inflammatory responses (Puré and Cuff 2001) and inflammation is a notable pathophysiological consequence following focal stroke (del Zoppo et al. 2000), one may speculate that the neuroprotective effect in CD44–/– mice over wild-type animals might be associated with its modulatory action on inflammatory responses to MCAO. This prediction has been supported by a number of previous studies using antileukocyte antibodies (Matsuo et al. 1994), antagonizing leukocyte adhesion molecules (e.g. intracellular adhesion molecule-1; Zhang et al. 1994), blocking inflammatory cytokines (e.g. the use of IL-1 receptor antagonist; Loddick and Rothwell 1996) and modulating inflammatory signaling pathways (e.g. p38 and ERK MAP kinases; Alessandrini et al. 1999; Barone et al. 2001).

Because CD44 was known to mediate signal transduction and induction of inflammatory gene expression in leukocytes and parenchymal cells (Puré and Cuff 2001), the expression of two key pro-inflammatory cytokines, IL-1β and TNFα, was measured in the CD44–/– and wild-type mice after MCAO. Interestingly, both cytokine mRNAs were induced in a similar level between CD44–/– and wild-type mice after MCAO, while the levels of soluble IL-1β protein were significantly lower in CD44–/– mice than in wild-type animals following MCAO. Since the current procedure used to detect IL-1β expression in the brain was optimized to measure the ‘soluble’ fraction, IL-1β protein detected by ELISA was likely to represent matured IL-1β, and the difference between IL-1β mRNA and its soluble protein expression in CD44–/– and wild-type mice might reflect its regulation at both post-transcriptional and post-translational levels.

Increase in IL-1β expression has been observed in several types of brain injury including excitotoxicity, LPS, brain trauma and ischemia (del Zoppo et al. 2000; Rothwell and Luheshi 2000). In particular, the significant up-regulation of IL-1β is recognized to play a detrimental role after brain ischemia since the blockade of IL-1 by interleukin-1 receptor antagonist was shown to be neuroprotective (Loddick and Rothwell 1996). Therefore, the suppression of IL-1β production in the ischemic brain tissue might be associated with the reduced ischemic damage in CD44–/– mice. In addition to its role in inflammation, IL-1β has multiple effects on neuronal, glial and endothelial cell function (Rothwell and Luheshi 2000) that may impede recovery following ischemic injury. On the other hand, however, it also should be pointed out that the interpretation of these data should be carried out with extreme caution as the changes in cytokine gene expression (such as IL-1β and TNFα) may be secondary to the reduced injury in CD44–/– mice following MCAO.

Additional mechanisms have been explored to understand the neuroprotective effects of CD44–/– mice after ischemic brain injury. As a robust expression of CD44 was identified in microglia of ischemic brain tissues (Wang et al. 2001), the temporal and spatial distribution of microglial activation (using the CD11b as a marker) was examined in the present study. However, no difference was observed between CD44–/– and wild-type mice after MCAO. Similarly, no difference was noted in gliosis (using GFAP immunostaining), apoptosis (measuring the levels of active caspase-3 and DNA fragmentation; data not shown), as well as the presence of various inflammatory cells in the ischemic tissues (using specific cellular markers for leukocytes, T-cells and dendritic cells; data not shown).

In conclusion, our present study provides direct evidence that CD44 expression following brain ischemia might play a role in ischemic brain injury since the deficiency of this gene resulted in a marked reduction in infarct size and neurological deficits. Similar to other inflammatory diseases, the role of CD44 in ischemic brain injury might be associated with the expression/function of some inflammatory cytokines such as IL-1β. Though we were not able to confirm a differential involvement in inflammatory cells following ischemic injury between CD44–/– and wild-type mice, the significant reduction of soluble IL-1β expression in CD44–/– mice and the substantial evidence on a detrimental role of IL-1β in such condition may suggest causal association.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Focal brain ischemia
  5. Neurological deficits and rota-rod test
  6. Physiological parameters
  7. Real-time RT-PCR
  8. Enzyme-linked immunosorbent assay for IL-1β
  9. Immunohistochemical analysis
  10. Statistical analysis
  11. Results
  12. Time-dependent expression of CD44 mRNA after transient MCAO in C57BL/6 mice
  13. CD44 deficiency protects brain from ischemic brain injury
  14. Effect on physiological parameters in CD44–/– mice after cerebral ischemia
  15. Cytokine gene expression in CD44–/– and wild-type mice after ischemic brain injury
  16. Activation of microglia and astrocytes in CD44–/– mice after ischemic brain injury
  17. Discussion
  18. References
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