Plasminogen activator inhibitor-1 mitigates brain injury in a rat model of infection-sensitized neonatal hypoxia-ischemia - PubMed (original) (raw)

Plasminogen activator inhibitor-1 mitigates brain injury in a rat model of infection-sensitized neonatal hypoxia-ischemia

Dianer Yang et al. Cereb Cortex. 2013 May.

Abstract

Intrauterine infection exacerbates neonatal hypoxic-ischemic (HI) brain injury and impairs the development of cerebral cortex. Here we used low-dose lipopolysaccharide (LPS) pre-exposure followed by unilateral cerebral HI insult in 7-day-old rats to study the pathogenic mechanisms. We found that LPS pre-exposure blocked the HI-induced proteolytic activity of tissue-type plasminogen activator (tPA), but significantly enhanced NF-κB signaling, microglia activation, and the production of pro-inflammatory cytokines in newborn brains. Remarkably, these pathogenic responses were all blocked by intracerebroventricular injection of a stable-mutant form of plasminogen activator protein-1 called CPAI. Similarly, LPS pre-exposure amplified, while CPAI therapy mitigated HI-induced blood-brain-barrier damage and the brain tissue loss with a therapeutic window at 4 h after the LPS/HI insult. The CPAI also blocks microglia activation following a brain injection of LPS, which requires the contribution by tPA, but not the urinary-type plasminogen activator (uPA), as shown by experiments in tPA-null and uPA-null mice. These results implicate the nonproteolytic tPA activity in LPS/HI-induced brain damage and microglia activation. Finally, the CPAI treatment protects near-normal motor and white matter development despite neonatal LPS/HI insult. Together, because CPAI blocks both proteolytic and nonproteolytic tPA neurotoxicity, it is a promising therapeutics of neonatal HI injury either with or without infection.

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Figures

Figure 1.

Figure 1.

CPAI prevents LPS/HI-induced BBB damage in immature brains. (A) Representative photographs of the brains from P8 rat pups treated with the indicated conditions (A1–A5) 24 h earlier and injected intraperitoenally with NaF at 2 h prior to sacrifice and transcardial perfusion (n = 4–5 as indicated in B). (B) Quantification of the NaF fluorescence from brain extracts. LPS/HI insults increased the extravascular NaF fluorescence to 461 ± 26% (mean ± SEM) of the baseline level in the carotid-occluded hemisphere (HI, asterisk in A). L, the opposite (left) side of brain; R, the right side of brain. *P < 0.01 by unpaired _t_-test. (C) Representative MMP zymogram at 24 h after the indicated treatment conditions (n = 4). Equal loading of brain extracts was verified by the immunoblot detection of β-actin. The optic density of MMP-9 bands was quantified (lower panel). The _P_-value was determined using unpaired _t_-test. (D) Representative immunoblotting against ZO-1 at 24 h after the indicated treatments (n = 3). Quantification (lower panel) showed that LPS/HI insults reduced the ZO-1 level to 36 ± 13% (mean ± SEM) of the baseline level, while CPAI treatment prevented the decrease of ZO-1 (98 ± 2%). P < 0.05 by unpaired _t_-test. Note that LPS administration by itself had little effects, but its addition to HI amplified NaF extravasation, MMP-9 activation, and ZO-1 reduction, which were all markedly mitigated by post-LPS/HI administration of CPAI.

Figure 2.

Figure 2.

CPAI prevents LPS/HI-induced NF-κB signaling activation in immature brains. (A) Immunoblot analysis of the distribution and clearance of CPAI injected unilaterally to the lateral ventricle (arrow) in P7 rat pups. R, right hemisphere; L, left hemisphere. Note the undetectable basal level of PAI-1 in the brain parenchyma, diffusion of injected CPAI in both hemispheres, and a rapid decline of the CPAI level at 2 h post-intracerebrventricular (ICV) injection. (B and C) Representative photographs of plasminogen–zymogram and immunoblot-detection of tPA, PAI-1, or β-actin from brain extracts of rat pups at 4 (B) or (C) 24 h after the indicated conditions (n = 5). +, positive-control lanes were loaded with recombinant tPA or CPAI. The optic density of tPA band at 4 h and uPA band at 24 h was quantified for each condition for comparison (lower panel). Note the strong inhibition of tPA activity at 4 h in saline- or CPAI-treated brains following LPS/HI insult, as well as the induction of uPA activity at 24 h in both HI- and LPS/HI-challenged brains without the CPAI treatment. (F) Immunoblot detection of IκBα from whole-cell lysates of the brains collected at 4 h after the indicated conditions (n = 3). L, the opposite/left hemisphere. R, the carotid-occluded (right) hemisphere. Quantification after normalized to β-actin (lower panel) showed that LPS/HI decreased IκBα to 55 ± 4% (mean ± SEM) of the basal level in unchallenged brains, while post-HI administration of CPAI almost completely prevented IκBα reduction (96 ± 1% of the baseline); P < 0.01 by unpaired _t_-test. (_G_) Representative NF-κB EMSA gel using the nuclear extract of brains collected at 4 h after indicated conditions (_n_ > 6). Mut, oligonucleotide probes with mutated NF-κB binding sequence. Note that the high-order protein–DNA complex was detected only in the sample from LPS/HI-injured brains, which was abolished by the CPAI treatment.

Figure 3.

Figure 3.

CPAI prevents LPS/HI-induced chemokine production and monocyte recruitment. (A) Representative results of a cytokine array using brain extracts at 24 h following indicated conditions (n = 2 for unchallenged and LPS-treated animals; n = 4 for LPS/HI-PBS or LPS/HI-CPAI treatments). The duplicated spots of MCP-1 and the quantification results were indicated. LPS/HI increased the brain MCP-1 level to 9.75 ± 0.54 (mean ± SD) fold of the baseline level, while CPAI treatment attenuated this induction. (B and C) ELISA analysis of the MCP-1 levels from brains (B) or the plasma (C) at 24 h after the indicated conditions (n = 4 for each). The MCP-1 level in the unchallenged brain was 4.4 ± 0.85 pg/mL extracts (mean ± SD), which was increased by LPS/HI insults to 50.3 ± 4.4 pg/mL (asterisk, P < 0.01) and significantly attenuated by the CPAI treatment (_P_ < 0.05 by unpaired _t_-test). In contrast, the basal plasma MCP-1 level was already high in unchallenged animals (116.7 ± 3.3 pg/mL), and upregulated by various stimuli to a similar degree. (_D_–_I_) Immunofluoresence detection of Iba1 (_D_, _E_, and _F_) and OX42/CD11b (_G_, _H_, and _I_) in coronal sections at the fornix-decussation level of brains collected at 16 h after pure-HI, LPS/HI-PBS, or LPS/HI-CPAI treatments (_n_> 5). White lines mark the boundaries of corpus callosum (CC). LV, lateral ventricles. Scale bar: 50 μm. Note that the CC in LPS/HI-PBS-treated rats was 2 times wider than that of LPS/HI-CPAI-treated rats and filled with numerous large, round Iba1/OX42 double-positive cells (248 ± 24 per visual field). In contrast, LPS/HI-CPAI-treated animals contained fewer Iba1+ cells within the corpus callosum (50 ± 11 per visual field) that exhibited ramified cytoplasmic processes and did not express OX42. Also note that the swelling of CC was milder in pure-HI-injured animal brains and there was no obvious OX42-immunoreactivity. (J) The tensor trace map from rat brain at 24 h after LPS/HI insult. Note the expansion/swelling of CC/external capsule on the HI-injured hemisphere (indicated by arrows).

Figure 4.

Figure 4.

CPAI mitigates LPS-induced and tPA-dependent cytokine production. (A and B) ELISA measurement of the TNFα and MCP-1 level in the brains of P8 rats at 24 h after intracerebroventricular injection of 1.9 μg LPS, as well as saline (PBS) or 1.9 μg CPAI injection on the opposite cerebral ventricle, normalized to unchallenged (UN) animals (n = 4 for each group). Note that ICV injection of LPS increased both TNFα (172 ± 2%; mean ± SD) and MCP-1 (1026 ± 150%) levels in the brain, which were significantly attenuated by CPAI treatment (96 ± 2% for TNFα and 612 ± 145% for MCP-1, P < 0.01 by unpaired _t_-test). (C and D) ELISA measurement of the TNFα and MCP-1 level in the brains of P10 wild-type, _tPA_-null, and _uPA_-null mice at 24 h after ICV injection of 20 μg LPS (n = 5 for unchallenged and LPS injection groups of each genotype). An LPS injection increased both TNFα (834 ± 88%; mean ± SE) and MCP-1 levels (297 ± 36%) in wild-type mice, but only mildly affected the expression of TNFα (336 ± 78%) and MCP-1 (104 ± 2%) in _tPA_-null mice. The increase of TNFα (724 ± 51%) and MCP-1 (269 ± 15%) levels in _uPA_-null mice was not significantly different from that in wild-type mice.

Figure 5.

Figure 5.

CPAI decreases LPS/HI-induced brain damage and impairment of motor functions. (A and B) Examples of the brains from rat pups receiving post-LPS/HI (10 min) ICV injection of saline (PBS, A) or 1.9 μg CPAI (B) at 7-day recovery. Note that the majority of saline-injected animals showed an obvious tissue loss on the right hemisphere (red arrows). (C) Quantification of tissue loss in the cerebral cortex, hippocampus, and striatum at 7 days after LPS/HI insult and ICV injection of saline (PBS) or 1.9 μg CPAI at 10 min, 2 h, or 4 h post-hypoxia. Shown are the percentages of tissue loss in each area to counterparts in contralateral hemisphere. With saline injection, there was a 43 ± 2.5% (mean ± SEM) tissue loss in the cerebral cortex, 44.2 ± 3.9% in the hippocampus, and 29.1 ± 3.3% in the striatum (n = 19). With CPAI injected at 10 min post-hypoxia (n = 20), the extent of tissue loss decreased to 4.7 ± 2.5% in the cerebral cortex, 9 ± 3.2% in the hippocampus, and 9.1 ± 2.8% in the striatum (n = 20). With CPAI injected at 2 h post-hypoxia, the tissue loss was 15 ± 2.6% in the cerebral cortex, 23.6 ± 4.6% in the hippocampus, and 9.1 ± 2.8% in the striatum (n = 17). With CPAI injection at 4 h post-hypoxia, the extent of tissue loss was 15.2 ± 4% in the cerebral cortex (P < 0.01 compared with saline injection by unpaired _t_-test), 28 ± 6.1% in the hippocampus (P < 0.05), and 9.1 ± 2.8% in the striatum (P = 0.07) (n = 18). (D) The body weight of unchallenged rat pups (n = 4) and those subjected to LPS/HI-insult at P7 followed by CPAI (n = 8) or saline (n = 9) treatment over 5-week recovery (P7–P42). Note that saline-treated rat pups initially gained body weight slower than unchallenged or CPAI-treated animals (asterisk: P < 0.05 by ANONA), but caught up in body weight after P28. (E) Comparison of the time (second) of staying on rotarods in unchallenged (UN, n = 4), LPS/HI-injured saline-injected (PBS, n = 9), and LPS/HI-injured CPAI-treated rat pups (CPAI, n = 8) when they reached 30 or 42 days of age. Note that the latency or falling from rotarods was significantly shorter in PBS-injected animals than that in CPAI-treated animals (P < 0.01 by unpaired _t_-test).

Figure 6.

Figure 6.

CPAI protects WM development of after neonatal LPS/HI injury. (A) Representative coronal (upper row) and transverse (lower row) images of DEC map of the brains from unchallenged (UN, n = 2), LPS/HI-PBS- (n = 3), or LPS/HI-CPAI-treated rat pups (n = 3) at 2 months of age. The directions of color-encoded water diffusion along x, y, and z axes in coronal/transverse views were indicated, respectively. Note that the DEC maps in unchallenged or LPS/HI-CPAI animals were bilaterally symmetric, while major axonal tracts in LPS/HI-PBS animals, especially ec, ic, and the fm were truncated, misplaced, or decreased in mass. MoDG, molecular layer of the dentate gyrus; opt, optic tract. (B) Comparison of FA, axial/longitudinal diffusivity (λll), and radial/transverse diffusivity (λ┴) in the fm by ex vivo DTI of 2-month-old animals under indicated conditions. Shown are the ratios of individual DTI parameters in the hemisphere ipsilateral to carotid-occlusion (R) and that in the contralateral hemisphere (L). The R/L ratio of FA values in LPS/HI saline-injected rats was reduced to 70 ± 6.2% (mean ± SD), while that in LPS/HI CPAI-treated animals was 96 ± 4.4%. The R/L ratio of λ┴ values was 208.6 ± 41.8% in LPS/HI saline-injected rats, compared with 108 ± 25% in CPAI-treated animals. Asterisks: P < 0.05 by ANOVA. (C) Comparison of DTI parameters in the external and internal capsule of unchallenged and LPS/HI CPAI-treated rats at 2 months of age. The R/L ratio of each DTI parameters was close to 1 in unchallenged and CPAI-treated animals.

Figure 7.

Figure 7.

Summary of how infection/LPS alters the pathogenic mechanisms of HI brain injury and how CPAI provides protection in both pure-HI and infection-sensitized HI insults through inhibition of tPA activities.

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