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Chronic intermittent hypoxia exerts CNS region-specific effects on rat microglial inflammatory and TLR4 gene expression.

Smith SM, Friedle SA, Watters JJ - PLoS ONE (2013)

Bottom Line: In the present studies, we tested the hypothesis that IH would differentially induce inflammatory factor gene expression in microglia in a CNS region-dependent manner, and that the effects of IH would differ temporally.Cortex, medulla and spinal cord tissues were dissected, microglia were immunomagnetically isolated and mRNA levels of the inflammatory genes iNOS, COX-2, TNFα, IL-1β and IL-6 and the innate immune receptor TLR4 were compared to levels in normoxia.We found that microglia from different CNS regions responded to IH differently.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin, United States of America.

ABSTRACT
Intermittent hypoxia (IH) during sleep is a hallmark of sleep apnea, causing significant neuronal apoptosis, and cognitive and behavioral deficits in CNS regions underlying memory processing and executive functions. IH-induced neuroinflammation is thought to contribute to cognitive deficits after IH. In the present studies, we tested the hypothesis that IH would differentially induce inflammatory factor gene expression in microglia in a CNS region-dependent manner, and that the effects of IH would differ temporally. To test this hypothesis, adult rats were exposed to intermittent hypoxia (2 min intervals of 10.5% O2) for 8 hours/day during their respective sleep cycles for 1, 3 or 14 days. Cortex, medulla and spinal cord tissues were dissected, microglia were immunomagnetically isolated and mRNA levels of the inflammatory genes iNOS, COX-2, TNFα, IL-1β and IL-6 and the innate immune receptor TLR4 were compared to levels in normoxia. Inflammatory gene expression was also assessed in tissue homogenates (containing all CNS cells). We found that microglia from different CNS regions responded to IH differently. Cortical microglia had longer lasting inflammatory gene expression whereas spinal microglial gene expression was rapid and transient. We also observed that inflammatory gene expression in microglia frequently differed from that in tissue homogenates from the same region, indicating that cells other than microglia also contribute to IH-induced neuroinflammation. Lastly, microglial TLR4 mRNA levels were strongly upregulated by IH in a region- and time-dependent manner, and the increase in TLR4 expression appeared to coincide with timing of peak inflammatory gene expression, suggesting that TLR4 may play a role in IH-induced neuroinflammation. Together, these data indicate that microglial-specific neuroinflammation may play distinct roles in the effects of intermittent hypoxia in different CNS regions.

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Intermittent hypoxia-induced inflammatory gene expression is rapid and transient in spinal microglia but sustained in spinal tissue homogenates.iNOS, COX-2, TNFα, IL-1β and IL-6 gene expression was analyzed by qRT-PCR in immunomagnetically-separated microglia (A) or tissue homogenates (B) from the spinal cord of healthy male rats exposed either to normoxia or IH for 1, 3 or 14 days. Means +/− 1 SEM are presented relative to expression in normoxic controls. *p<0.05; **p<0.01; ***p<0.001; #p = 0.074.
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pone-0081584-g003: Intermittent hypoxia-induced inflammatory gene expression is rapid and transient in spinal microglia but sustained in spinal tissue homogenates.iNOS, COX-2, TNFα, IL-1β and IL-6 gene expression was analyzed by qRT-PCR in immunomagnetically-separated microglia (A) or tissue homogenates (B) from the spinal cord of healthy male rats exposed either to normoxia or IH for 1, 3 or 14 days. Means +/− 1 SEM are presented relative to expression in normoxic controls. *p<0.05; **p<0.01; ***p<0.001; #p = 0.074.

Mentions: IL-1β was the only inflammatory gene to exhibit a strong upregulation within 1 day of IH in spinal microglia (p = <0.001) (Fig. 3A). However, by 3 days of IH, iNOS, COX-2, IL-1β and IL-6 mRNA levels were increased by 3–5 fold, but only COX-2 (p = 0.007) and IL-1β (p<0.001) levels attained statistical significance (iNOS p =  0.12; TNFα p =  0.28; IL-6 p =  0.099). COX-2 and IL-1β expression returned to basal levels by 14 days of IH exposure. IH did not appear to increase microglial IL-6 mRNA levels until 14 days although this 3-fold change was not statistically significant (p = 0.099). Spinal homogenates behaved very similarly to brainstem homogenates where the expression of all inflammatory genes examined was increased between 3 and 8-fold at 3 days of IH (Fig. 3B). COX-2 and IL-1β expression remained elevated at 14 days of IH (p = 0.003 and p = 0.033 respectively), but iNOS mRNA levels were returning to baseline despite remaining significantly elevated above basal levels (p = 0.044). Also, as in medullary homogenates, spinal homogenate IL-6 mRNA levels were highest at 14 days of IH (p = <0.001) while TNFα mRNA levels appeared to peak at 3 days (though not statistically significantly; p = 0.074).


Chronic intermittent hypoxia exerts CNS region-specific effects on rat microglial inflammatory and TLR4 gene expression.

Smith SM, Friedle SA, Watters JJ - PLoS ONE (2013)

Intermittent hypoxia-induced inflammatory gene expression is rapid and transient in spinal microglia but sustained in spinal tissue homogenates.iNOS, COX-2, TNFα, IL-1β and IL-6 gene expression was analyzed by qRT-PCR in immunomagnetically-separated microglia (A) or tissue homogenates (B) from the spinal cord of healthy male rats exposed either to normoxia or IH for 1, 3 or 14 days. Means +/− 1 SEM are presented relative to expression in normoxic controls. *p<0.05; **p<0.01; ***p<0.001; #p = 0.074.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3852519&req=5

pone-0081584-g003: Intermittent hypoxia-induced inflammatory gene expression is rapid and transient in spinal microglia but sustained in spinal tissue homogenates.iNOS, COX-2, TNFα, IL-1β and IL-6 gene expression was analyzed by qRT-PCR in immunomagnetically-separated microglia (A) or tissue homogenates (B) from the spinal cord of healthy male rats exposed either to normoxia or IH for 1, 3 or 14 days. Means +/− 1 SEM are presented relative to expression in normoxic controls. *p<0.05; **p<0.01; ***p<0.001; #p = 0.074.
Mentions: IL-1β was the only inflammatory gene to exhibit a strong upregulation within 1 day of IH in spinal microglia (p = <0.001) (Fig. 3A). However, by 3 days of IH, iNOS, COX-2, IL-1β and IL-6 mRNA levels were increased by 3–5 fold, but only COX-2 (p = 0.007) and IL-1β (p<0.001) levels attained statistical significance (iNOS p =  0.12; TNFα p =  0.28; IL-6 p =  0.099). COX-2 and IL-1β expression returned to basal levels by 14 days of IH exposure. IH did not appear to increase microglial IL-6 mRNA levels until 14 days although this 3-fold change was not statistically significant (p = 0.099). Spinal homogenates behaved very similarly to brainstem homogenates where the expression of all inflammatory genes examined was increased between 3 and 8-fold at 3 days of IH (Fig. 3B). COX-2 and IL-1β expression remained elevated at 14 days of IH (p = 0.003 and p = 0.033 respectively), but iNOS mRNA levels were returning to baseline despite remaining significantly elevated above basal levels (p = 0.044). Also, as in medullary homogenates, spinal homogenate IL-6 mRNA levels were highest at 14 days of IH (p = <0.001) while TNFα mRNA levels appeared to peak at 3 days (though not statistically significantly; p = 0.074).

Bottom Line: In the present studies, we tested the hypothesis that IH would differentially induce inflammatory factor gene expression in microglia in a CNS region-dependent manner, and that the effects of IH would differ temporally.Cortex, medulla and spinal cord tissues were dissected, microglia were immunomagnetically isolated and mRNA levels of the inflammatory genes iNOS, COX-2, TNFα, IL-1β and IL-6 and the innate immune receptor TLR4 were compared to levels in normoxia.We found that microglia from different CNS regions responded to IH differently.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin, United States of America.

ABSTRACT
Intermittent hypoxia (IH) during sleep is a hallmark of sleep apnea, causing significant neuronal apoptosis, and cognitive and behavioral deficits in CNS regions underlying memory processing and executive functions. IH-induced neuroinflammation is thought to contribute to cognitive deficits after IH. In the present studies, we tested the hypothesis that IH would differentially induce inflammatory factor gene expression in microglia in a CNS region-dependent manner, and that the effects of IH would differ temporally. To test this hypothesis, adult rats were exposed to intermittent hypoxia (2 min intervals of 10.5% O2) for 8 hours/day during their respective sleep cycles for 1, 3 or 14 days. Cortex, medulla and spinal cord tissues were dissected, microglia were immunomagnetically isolated and mRNA levels of the inflammatory genes iNOS, COX-2, TNFα, IL-1β and IL-6 and the innate immune receptor TLR4 were compared to levels in normoxia. Inflammatory gene expression was also assessed in tissue homogenates (containing all CNS cells). We found that microglia from different CNS regions responded to IH differently. Cortical microglia had longer lasting inflammatory gene expression whereas spinal microglial gene expression was rapid and transient. We also observed that inflammatory gene expression in microglia frequently differed from that in tissue homogenates from the same region, indicating that cells other than microglia also contribute to IH-induced neuroinflammation. Lastly, microglial TLR4 mRNA levels were strongly upregulated by IH in a region- and time-dependent manner, and the increase in TLR4 expression appeared to coincide with timing of peak inflammatory gene expression, suggesting that TLR4 may play a role in IH-induced neuroinflammation. Together, these data indicate that microglial-specific neuroinflammation may play distinct roles in the effects of intermittent hypoxia in different CNS regions.

Show MeSH
Related in: MedlinePlus