Assessment of the Therapeutic Potential of Metallothionein-II Application in Focal Cerebral Ischemia In Vitro and In Vivo

Abstract

Metallothionein-II (MT-II) is an ubiquitously expressed small-molecular-weight protein and highly induced in various species and tissues upon stress, inflammation, and ischemia. MT-deficiency exacerbates ischemic injury in rodent stroke models in vitro and in vivo. However, there is conflicting data on the potential neuroprotective effect of exogenously applied metallothionein. Thus, we applied MT-II in an in vitro stroke model and intraperitoneally (i.p.) in two in vivo standard models of transient middle cerebral artery occlusion (MCAO) (a 'stringent' one [60 min MCAO/48 h reperfusion] and a 'mild' one [30 min MCAO/72 h reperfusion]), as well as i.v. together with recombinant tissue plasminogen activator (rtPA) to evaluate if exogenous MT-II-application protects against ischemic stroke. Whereas MT-II did not protect against 60 min MCAO, there was a significant reduction of direct and indirect infarct volumes and neurological deficit in the MT-II (i.p.) treated animals in the 'mild' model at 3d after MCAO. Furthermore, MT-II also improved survival of the mice after MCAO, suppressed TNF-α mRNA induction in ischemic brain tissue, and protected primary neuronal cells against oxygen-glucose-deprivation in vitro. Thus, exogenous application of MT-II protects against ischemic injury in vitro and in vivo. However, long-term studies with different species and larger sampling sizes are required before a clinical use can be envisaged.

Fig 1. Primary neuronal cells treated with metallothionein-II (MT-II) are protected against oxygen glucose deprivation (OGD) in vitro.

Shown is the relative LDH release of primary neuronal cells after OGD (shown as “cell death (%LDH release)”). Cells are treated with various different concentrations of MT-II before induction of oxygen and glucose deprivation (MT-II: primary neuronal cells pretreated with metallothionein-II 12 h before OGD; co: control cells without OGD [BSS₂₀]). (*p = 0.0198, as calculated by Mann Whitney test; one-tailed).

Fig 2. Effect of metallothionein-II i.p. treatment in vivo 48h after MCAO for 60 min.

Infarct volumes of vehicle (0.9% NaCl) (n = 7) or MT-II (i.p.) treated (n = 8) male adult wildtype C57BL/6N mice after 60min MCAO and 48h reperfusion. A: direct infarct volumes. B: inflammatory cell count in the ischemic hemisphere as determined by counting Iba1-positive cells in whole ischemic brain hemispheres of mice at 48h of reperfusion after 60min MCAO. Inflammatory cell accumulation (macrophages and activated microglia) was determined by counting Iba1-positive cells at interaural position No.III (distance to bregma 3.9mm) in the whole ischemic/ipsilateral hemisphere. C: Brain swelling, as calculated by the difference between direct and indirect infarct volumes. Whereas indirect infarct volumes were calculated as the volume of the contralateral hemisphere minus the non-infarcted volume of the ipsilateral/ischemic hemisphere (p-values between groups were >0.05 as calculated by Mann Whitney U-Test, one-tailed). D: neurological deficit of mice at 48h after induction of MCAO as determined by a modified Bederson score [41]: 0 represents no deficits, 1 represents an extension deficit in the contralateral leg, 2 a hemiparesis with circling, 3 loss of postural reflexes, and 4 death.

Fig 3. Effect of metallothionein-II i.p. treatment in vivo 72h after MCAO for 30 min.

Infarct volumes of vehicle (0.9% NaCl) (n = 8) or MT-II i.p. (n = 9) treated male adult wildtype C57BL/6N mice after 60min MCAO and 48h reperfusion. A: Direct infarct volumes (*p-value < 0.05 as calculated by Mann Whitney U-Test, one-tailed). B: Inflammatory cell count in the ischemic hemisphere as determined by counting Iba1-positive cells in whole ischemic brain hemispheres of mice at 72h of reperfusion after 30min MCAO. Inflammatory cell accumulation (macrophages and activated microglia) was determined by counting Iba1-positive cells at interaural position No.III (distance to bregma 3.9mm) in the whole ischemic/ipsilateral hemisphere C: Brain swelling, as calculated by the difference between direct and indirect infarct volumes. Whereas indirect infarct volumes were calculated as the volume of the contralateral hemisphere minus the non-infarcted volume of the ipsilateral/ischemic hemisphere. (NaCl: vehicle-treated mice, MT-II: MT-II i.p.-treated wild-type mice). D: Neurological deficit of mice at 72h after induction of MCAO as determined by a modified Bederson score [41]: 0 represents no deficits, 1 represents an extension deficit in the contralateral leg, 2 a hemiparesis with circling, 3 loss of postural reflexes, and 4 death (*p < 0.05).

Fig 4. Effect of i.v. metallothionein-II treatment in vivo evaluated at 48h after MCAO for 45 min.

Infarct volumes of vehicle (0.9% NaCl) (n = 8), MT-II (i.v.) (n = 12), rtPA (i.v.) (n = 11), or rtPA and MT-II co-application (i.v.) (n = 11) injected wildtype C57BL6/N male mice after 45min MCAO and 48h (A), Kaplan-Meier Analysis of animal survival (B), and brain swelling (C; as calculated by the difference of direct and indirect infarct volumes), and neurological deficit (D) in a standard model of cerebral ischemia (45min MCAO and 48h reperfusion) after intravenous treatment with metallothionein-II or vehicle only in combination with or without co-application of rtPA (*p = 0.0187; as calculated by log rank Mantel Cox test). Data A presented as scatter dot blots in combination with mean ± SEM. For comparison of infarct volumes and neurological deficit, ANOVA with Kruskal-Wallis test was used.

Fig 5. Evaluation by the use of immunohistofluorescence reveals no difference between MT-II and NaCl treated mice 48h after induction of MCAO.

Neurons, as well as macrophages/microglia were stained by the use of anti-NeuN- (in red) and anti-Iba1-specific antibodies (in green) in brain slices derived at 48h reperfusion after induction of cerebral ischemia in adult male C57BL/6N wild-type mice, treated with NaCl only (A), or MT-II (5μg MT-II/10g body weight i.v. at start of reperfusion) (B). As shown in representative figures, neuron-specific staining (red) showed no significant alteration in ipsilateral (c,d,i,j) or contralateral/non-ischemic hemisphere after MT-II treatment when compared to NaCl-only treatment after MCAO (f,g,l,m). Moreover, Iba1-specific staining (for macrophages/microglia) does not reveal a clearly visible alteration after MT-II treatment (k,n) when compared to NaCl-only treatment (e,h) after MCAO.

Fig 6. Gene expression analysis of pro-inflammatory proteins with real-time PCR.

Relative mRNA expression of selected pro-inflammatory proteins was analysed in real-time PCR with gene-specific primers in whole mouse brain hemispheres of adult male wild-type C57BL/6N mice treated with either MT-II i.p. only without MCAO (MT-II-treated; n = 3), in mice at 72h after 30min MCAO that were treated with MT-II i.p. (n = 4), and in mice at 72h after 30min MCAO that were treated with vehicle only (n = 6). cDNA was separately isolated from each hemisphere. Relative Expression is presented as fold changes, calculated with the ΔΔCt-Method, normalized to HPRT mRNA expression and related to the non-ischemic/contralateral hemispheres as references. TNFα induction after MCAO is significantly reduced in mice that were treated with MT-II i.p when compared to vehicle-only treated mice (*p < 0.05, students one-side t-test). Data are shown as relative mRNA expression of each gene and is calculated from two technical replications of n = 3 to 6 mice for each condition (values are given as mean ± SEM).