Inhibition of gelatinase activity reduces neural injury in an ex vivo model of hypoxia-ischemia

Abstract

Perinatal hypoxia-ischemia (H-I) often manifests as cognitive and/or motor disturbances that appear early in development. Growing evidence indicates that neuroinflammation may exacerbate H-I injury. Resident microglia release proinflammatory cytokines and proteases in response to ischemia. Matrix metalloproteinases (MMPs), in particular, activate cytokines and degrade basement membrane proteins. These actions ultimately permit entry of peripheral leukocytes into the CNS neuropil, enhancing neuroinflammation and cell death. Currently, the relative contributions of resident and peripheral immune cells to ischemic brain injury are unclear. The present study employed an ex vivo model of H-I through oxygen glucose deprivation (OGD) to identify the cellular localization of MMP-9 in organotypic hippocampal slices from rat, and to determine whether inhibiting gelatin-degrading MMPs affords neuroprotection in the absence of peripheral immune cells. Immunohistochemistry revealed ubiquitous neuronal MMP-9 expression in both normoxic and hypoxic slices. Increased MMP-9 expression was detected in CD11b-positive microglia after 48 h exposure to OGD relative to normoxic controls. Consistent with these data, in situ zymography showed increased gelatinolytic activity after OGD. Gelatin-cleaved fluorescence localized to astrocytic processes and somata of various cellular morphologies. Treatment with either the MMP inhibitor AG3340 (prinomastat) or minocycline dampened OGD-induced gelatinolytic activity and neural injury, as measured by Fluoro-Jade staining, relative to vehicle controls. These results show that resident microglia, in the absence of peripheral immune cells, were sufficient to enhance neural injury after OGD in the organotypic hippocampal slice. Additionally, these effects were associated with upregulation or secretion of MMP-9, and were blocked after treatment with either the gelatinase-selective compound AG3340 or the anti-inflammatory compound minocycline. These data, coupled with the effectiveness of these compounds previously shown in vivo, support the selective targeting of gelatin-degrading MMPs and activated microglia as potential therapeutic approaches to combat neonatal H-I injury.

Figure 1. OGD in the organotypic hippocampal slice

Organotypic slices were cultured for 14 days and exposed to 48 hrs of normoxia or OGD. Thionin staining revealed intact hippocampal slices (A, inset) with dense neuronal layers (A, dark labeling) after exposure to normoxia. Fluoro-Jade staining revealed neural injury that was predominantly localized to the neuronal layers after OGD (B, white labeling), demonstrating selectivity for neuronal cell death. OGD – induced cell death was validated with propidium iodide (PI) staining (C,D, white labeling), showing marked PI elevations in slices exposed to OGD (D) compared to normoxic controls (C). Scale bars = 100 μm.

Figure 2. Verification of gelatinase inhibition and MMP-9 specificity

In situ zymography (A,B,D,E) was performed in tissues from rats that received 10 μg cortical infusions of LPS (B, arrow). Gelatin-cleaved fluorescence was prominent in cortical regions adjacent to the LPS infusion site (B,D). Fluorescent signal in contralateral control hemispheres was faint by comparison and was detected only in blood vessels (A,D). Consistent with increased gelatinase activity, LPS induced elevations in CD-45 (C) and MMP-9 (F) immunoreactivity. Gelatin zymography was performed using 2 ng and 4 ng of purified gelatinase zymography standards (G, left panel). Intense bands of cleaved gelatin were detected at the predicted positions on SDS-PAGE for MMP-9 (92 kDa) and MMP-2 (68, 62 kDa). Incubation with 3 mM AG3340 showed complete inhibition of gelatinase activity. Western Blot of cortical homogenates from ischemic rat brain (G, right panel) shows a single band migrated at the predicted position on SDS-PAGE for activated MMP-9 (∼78 kDa). N = 2 (in situ zymography), N = 4 (Western Blot; R = ischemic hemisphere). Scale bars = 100 μm. Arrows indicate cells expressing both antigens.

Figure 3. Basal MMP-9 expression

Micrographs show cellular expression of MMP-9 (A,D,H), GFAP (B), CD11b (E) and NSE (I) after 48 hrs exposure to normoxia. (A-C) Basal MMP-9 expression colocalized with astrocytic processes (C). (D-F) CD11b – expressing microglia displayed the activated phenotype in normoxic slices (E) but did not colocalize with MMP-9 (F). (G-J) MMP-9 expression was prominent on cell surfaces of NSE-immunopositive neurons (J). Scale bars = 100 μm. Arrows indicate cells expressing both antigens.

Figure 4. MMP-9 expression after OGD

Micrographs show cellular expression of MMP-9 (B,E,I), GFAP (A), CD11b (D) and NSE (H) after 48 hrs exposure to OGD. (A-C) Hypertrophic astrocytes showed intense GFAP immunoreactivity (A) but little colocalization with MMP-9 (C). (D-F) CD11b – expressing microglia were ubiquitous (D) and colocalized with MMP-9 (F) after OGD. (G-J) NSE – positive neurons (H) expressed MMP-9 on cell surfaces (J). Scale bars = 100 μm. Arrows indicate cells expressing both antigens.

Figure 5. Gelatinase activity is elevated after OGD

Confocal micrographs show in situ zymography in organotypic hippocampal slices after 48 hrs exposure to normoxia or OGD. Basal gelatinase activity was detected in normoxic cells (A,C) and was increased after exposure to OGD (B,D). Gelatinase activity localized to cellular processes and cells with amoeboid morphology after OGD (D) when compared to normoxia (C). Scale bars = 100 μm (A,B), 50 μm (C,D).

Figure 6. Treatment with AG3340 or minocycline reduces gelatinase activity

Gelatin-cleaved fluorescence was quantified in organotypic hippocampal slices after exposure to normoxia or OGD in media containing vehicle, AG3340, or minocycline. Slices treated with vehicle alone showed a significant increase in fluorescent signal after exposure to OGD relative to normoxic controls (p<0.01). Gelatinase activity was significantly reduced in slices exposed to OGD and treated with either AG3340 (p<0.01) or minocycline (p<0.05) relative to those treated with vehicle alone. N = 6. * indicates significant from normoxia; # indicates significant from OGD + vehicle.

Figure 7. Treatment with AG3340 or minocycline reduces neural injury

Organotypic hippocampal slices exposed to normoxia or OGD in media containing vehicle, AG3340, or minocycline were stained with Fluoro-Jade. Basal Fluoro-Jade staining was detected in slices exposed to normoxia after all treatments (A-C). Staining was markedly elevated in slices exposed to OGD and treated with vehicle alone (D) compared to normoxic controls (A), and this effect was attenuated in slices exposed to OGD and treated with either AG3340 (E) or minocycline (F) (scale bars = 100 μm). Quantification (G) showed a significant increase in slices exposed to OGD and treated with vehicle alone relative to normoxic controls (p<0.001). Treatment with either AG3340 or minocycline attenuated the OGD – induced increase in Fluoro-Jade stain (p<0.001), and minocycline reduced staining to normoxic control levels. N = 6. * indicates significant from normoxia; # indicates significant from OGD + vehicle.