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Parecoxib is neuroprotective in spontaneously hypertensive rats after transient middle cerebral artery occlusion: a divided treatment response?

Kelsen J, Kjaer K, Chen G, Pedersen M, Røhl L, Frøkiaer J, Nielsen S, Nyengaard JR, Rønn LC - J Neuroinflammation (2006)

Bottom Line: We found a significant reduction in infarct volume in parecoxib treated animals one week after tMCAo (p < 0.03).We found indications of mRNA up-regulation of IL-1beta, IL-6, TNF-alpha and COX-2, whereas COX-1 remained unaffected.IP parecoxib administration during tMCAo was neuroprotective, as evidenced by a large reduction in mean infarct volume and a lower cortical ADC increment.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Water and Salt Research Centre, University of Aarhus, DK-8000 Aarhus C, Denmark. jesper.kelsen@ki.au.dk

ABSTRACT

Background: Anti-inflammatory treatment affects ischemic damage and neurogenesis in rodent models of cerebral ischemia. We investigated the potential benefit of COX-2 inhibition with parecoxib in spontaneously hypertensive rats (SHRs) subjected to transient middle cerebral artery occlusion (tMCAo).

Methods: Sixty-four male SHRs were randomized to 90 min of intraluminal tMCAo or sham surgery. Parecoxib (10 mg/kg) or isotonic saline was administered intraperitoneally (IP) during the procedure, and twice daily thereafter. Nineteen animals were euthanized after 24 hours, and each hemisphere was examined for mRNA expression of pro-inflammatory cytokines and COX enzymes by quantitative RT-PCR. Twenty-three tMCAo animals were studied with diffusion and T2 weighted MRI within the first 24 hours, and ten of the SHRs underwent follow-up MRI six days later. Thirty-three SHRs were given 5-bromo-2'-deoxy-uridine (BrdU) twice daily on Day 4 to 7 after tMCAo. Animals were euthanized on Day 8 and the brains were studied with free-floating immunohistochemistry for activated microglia (ED-1), hippocampal granule cell BrdU incorporation, and neuronal nuclei (NeuN). Infarct volume estimation was done using the 2D nucleator and Cavalieri principle on NeuN-stained coronal brain sections. The total number of BrdU+ cells in the dentate gyrus (DG) of the hippocampus was estimated using the optical fractionator.

Results: We found a significant reduction in infarct volume in parecoxib treated animals one week after tMCAo (p < 0.03). Cortical ADC values in the parecoxib group were markedly less increased on Day 8 (p < 0.01). Interestingly, the parecoxib treated rats were segregated into two subgroups, suggesting a responder vs. non-responder phenomenon. We found indications of mRNA up-regulation of IL-1beta, IL-6, TNF-alpha and COX-2, whereas COX-1 remained unaffected. Hippocampal granule cell BrdU incorporation was not affected by parecoxib treatment. Presence of ED-1+ activated microglia in the hippocampus was related to an increase in BrdU uptake in the DG.

Conclusion: IP parecoxib administration during tMCAo was neuroprotective, as evidenced by a large reduction in mean infarct volume and a lower cortical ADC increment. Increased pro-inflammatory cytokine mRNA levels and hippocampal granule cell BrdU incorporation remained unaffected.

No MeSH data available.


Related in: MedlinePlus

COX-2 and NeuN double stains 24 hours and one week after tMCAo. The COX-2 IHC was developed with nickel-enhanced DAB (black), whereas NeuN was visualized with NovaRed® (brownish red). The images 5A and 5B are obtained from a pilot study where the animal was euthanized 24 hours after tMCAo. 5A visualizes a relatively small neocortical infarct in the right hemisphere. The box delineates a part of the ischemic border zone that is shown at forty times magnification in 5B. The penumbra contains large swollen neurons that express the membrane-bound COX-2 enzyme. In the infarct core the neurons tend to be small and star-shaped due to irreversible neuronal death. 5C and 5E are from a saline-treated animal one week after tMCAo. Forty times magnifications of the boxes are shown in 5D and 5F. The neurons in the border zone on Day 8 after ischemic injury showed a perinuclear expression pattern of the COX-2 enzyme (5D). COX-2+ neurons can be found in areas like the neocortex, piriform cortex and the DG of the hippocampus under normal conditions. 5F shows COX-2 expressed in dendrites of neurons in the molecular cell layer of the DG. The scale bar in 5A is 5 mm, whereas the scale bars in 5B, 5D and 5F equals 50 μm.
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Figure 5: COX-2 and NeuN double stains 24 hours and one week after tMCAo. The COX-2 IHC was developed with nickel-enhanced DAB (black), whereas NeuN was visualized with NovaRed® (brownish red). The images 5A and 5B are obtained from a pilot study where the animal was euthanized 24 hours after tMCAo. 5A visualizes a relatively small neocortical infarct in the right hemisphere. The box delineates a part of the ischemic border zone that is shown at forty times magnification in 5B. The penumbra contains large swollen neurons that express the membrane-bound COX-2 enzyme. In the infarct core the neurons tend to be small and star-shaped due to irreversible neuronal death. 5C and 5E are from a saline-treated animal one week after tMCAo. Forty times magnifications of the boxes are shown in 5D and 5F. The neurons in the border zone on Day 8 after ischemic injury showed a perinuclear expression pattern of the COX-2 enzyme (5D). COX-2+ neurons can be found in areas like the neocortex, piriform cortex and the DG of the hippocampus under normal conditions. 5F shows COX-2 expressed in dendrites of neurons in the molecular cell layer of the DG. The scale bar in 5A is 5 mm, whereas the scale bars in 5B, 5D and 5F equals 50 μm.

Mentions: As shown in Figure 4A, the COX-1 mRNA expression was not affected by transient focal brain ischemia. On the contrary, we found clear indications of a higher COX-2 mRNA level 24 hours after ischemia (Figure 4B). The COX-2 up-regulation happened regardless of whether the animals underwent saline or parecoxib treatment. In immunohistochemical pilot studies we found a consistent COX-2 protein presence in the border zone of the infarct 24 hours after tMCAo (Figure 5A and 5B). On Day 8 after ischemia it was impossible to visualize the same COX-2 protein expression around the matured infarct (Figure 5C and 5D). Together with IL-1β and IL-6, TNF-α is one of the major pro-inflammatory cytokines released by activated microglia following ischemic brain injury. For all three cytokines, we saw a similar mRNA expression pattern 24 hours after tMCAo (Figure 4C, 4D, and 4E). In the two ischemia groups our measurements indicated an mRNA up-regulation of TNF-α, IL-1β, and IL-6 that was unaffected by COX-2 enzyme blockage. For TNF-α, the mRNA up-regulation differed significantly between the parecoxib-treated tMCAo group and the two sham groups. The significant differences in TNF-α expression should be interpreted with caution due to large spreads in small sample sizes.


Parecoxib is neuroprotective in spontaneously hypertensive rats after transient middle cerebral artery occlusion: a divided treatment response?

Kelsen J, Kjaer K, Chen G, Pedersen M, Røhl L, Frøkiaer J, Nielsen S, Nyengaard JR, Rønn LC - J Neuroinflammation (2006)

COX-2 and NeuN double stains 24 hours and one week after tMCAo. The COX-2 IHC was developed with nickel-enhanced DAB (black), whereas NeuN was visualized with NovaRed® (brownish red). The images 5A and 5B are obtained from a pilot study where the animal was euthanized 24 hours after tMCAo. 5A visualizes a relatively small neocortical infarct in the right hemisphere. The box delineates a part of the ischemic border zone that is shown at forty times magnification in 5B. The penumbra contains large swollen neurons that express the membrane-bound COX-2 enzyme. In the infarct core the neurons tend to be small and star-shaped due to irreversible neuronal death. 5C and 5E are from a saline-treated animal one week after tMCAo. Forty times magnifications of the boxes are shown in 5D and 5F. The neurons in the border zone on Day 8 after ischemic injury showed a perinuclear expression pattern of the COX-2 enzyme (5D). COX-2+ neurons can be found in areas like the neocortex, piriform cortex and the DG of the hippocampus under normal conditions. 5F shows COX-2 expressed in dendrites of neurons in the molecular cell layer of the DG. The scale bar in 5A is 5 mm, whereas the scale bars in 5B, 5D and 5F equals 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1764728&req=5

Figure 5: COX-2 and NeuN double stains 24 hours and one week after tMCAo. The COX-2 IHC was developed with nickel-enhanced DAB (black), whereas NeuN was visualized with NovaRed® (brownish red). The images 5A and 5B are obtained from a pilot study where the animal was euthanized 24 hours after tMCAo. 5A visualizes a relatively small neocortical infarct in the right hemisphere. The box delineates a part of the ischemic border zone that is shown at forty times magnification in 5B. The penumbra contains large swollen neurons that express the membrane-bound COX-2 enzyme. In the infarct core the neurons tend to be small and star-shaped due to irreversible neuronal death. 5C and 5E are from a saline-treated animal one week after tMCAo. Forty times magnifications of the boxes are shown in 5D and 5F. The neurons in the border zone on Day 8 after ischemic injury showed a perinuclear expression pattern of the COX-2 enzyme (5D). COX-2+ neurons can be found in areas like the neocortex, piriform cortex and the DG of the hippocampus under normal conditions. 5F shows COX-2 expressed in dendrites of neurons in the molecular cell layer of the DG. The scale bar in 5A is 5 mm, whereas the scale bars in 5B, 5D and 5F equals 50 μm.
Mentions: As shown in Figure 4A, the COX-1 mRNA expression was not affected by transient focal brain ischemia. On the contrary, we found clear indications of a higher COX-2 mRNA level 24 hours after ischemia (Figure 4B). The COX-2 up-regulation happened regardless of whether the animals underwent saline or parecoxib treatment. In immunohistochemical pilot studies we found a consistent COX-2 protein presence in the border zone of the infarct 24 hours after tMCAo (Figure 5A and 5B). On Day 8 after ischemia it was impossible to visualize the same COX-2 protein expression around the matured infarct (Figure 5C and 5D). Together with IL-1β and IL-6, TNF-α is one of the major pro-inflammatory cytokines released by activated microglia following ischemic brain injury. For all three cytokines, we saw a similar mRNA expression pattern 24 hours after tMCAo (Figure 4C, 4D, and 4E). In the two ischemia groups our measurements indicated an mRNA up-regulation of TNF-α, IL-1β, and IL-6 that was unaffected by COX-2 enzyme blockage. For TNF-α, the mRNA up-regulation differed significantly between the parecoxib-treated tMCAo group and the two sham groups. The significant differences in TNF-α expression should be interpreted with caution due to large spreads in small sample sizes.

Bottom Line: We found a significant reduction in infarct volume in parecoxib treated animals one week after tMCAo (p < 0.03).We found indications of mRNA up-regulation of IL-1beta, IL-6, TNF-alpha and COX-2, whereas COX-1 remained unaffected.IP parecoxib administration during tMCAo was neuroprotective, as evidenced by a large reduction in mean infarct volume and a lower cortical ADC increment.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Water and Salt Research Centre, University of Aarhus, DK-8000 Aarhus C, Denmark. jesper.kelsen@ki.au.dk

ABSTRACT

Background: Anti-inflammatory treatment affects ischemic damage and neurogenesis in rodent models of cerebral ischemia. We investigated the potential benefit of COX-2 inhibition with parecoxib in spontaneously hypertensive rats (SHRs) subjected to transient middle cerebral artery occlusion (tMCAo).

Methods: Sixty-four male SHRs were randomized to 90 min of intraluminal tMCAo or sham surgery. Parecoxib (10 mg/kg) or isotonic saline was administered intraperitoneally (IP) during the procedure, and twice daily thereafter. Nineteen animals were euthanized after 24 hours, and each hemisphere was examined for mRNA expression of pro-inflammatory cytokines and COX enzymes by quantitative RT-PCR. Twenty-three tMCAo animals were studied with diffusion and T2 weighted MRI within the first 24 hours, and ten of the SHRs underwent follow-up MRI six days later. Thirty-three SHRs were given 5-bromo-2'-deoxy-uridine (BrdU) twice daily on Day 4 to 7 after tMCAo. Animals were euthanized on Day 8 and the brains were studied with free-floating immunohistochemistry for activated microglia (ED-1), hippocampal granule cell BrdU incorporation, and neuronal nuclei (NeuN). Infarct volume estimation was done using the 2D nucleator and Cavalieri principle on NeuN-stained coronal brain sections. The total number of BrdU+ cells in the dentate gyrus (DG) of the hippocampus was estimated using the optical fractionator.

Results: We found a significant reduction in infarct volume in parecoxib treated animals one week after tMCAo (p < 0.03). Cortical ADC values in the parecoxib group were markedly less increased on Day 8 (p < 0.01). Interestingly, the parecoxib treated rats were segregated into two subgroups, suggesting a responder vs. non-responder phenomenon. We found indications of mRNA up-regulation of IL-1beta, IL-6, TNF-alpha and COX-2, whereas COX-1 remained unaffected. Hippocampal granule cell BrdU incorporation was not affected by parecoxib treatment. Presence of ED-1+ activated microglia in the hippocampus was related to an increase in BrdU uptake in the DG.

Conclusion: IP parecoxib administration during tMCAo was neuroprotective, as evidenced by a large reduction in mean infarct volume and a lower cortical ADC increment. Increased pro-inflammatory cytokine mRNA levels and hippocampal granule cell BrdU incorporation remained unaffected.

No MeSH data available.


Related in: MedlinePlus