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. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: Stroke. 2018 Mar 6;49(4):995–1002. doi: 10.1161/STROKEAHA.117.019860

Minocycline Effects on Intracerebral Hemorrhage-Induced Iron Overload in Aged Rats: Brain Iron Quantification with MRI

Shenglong Cao 1,2, Ya Hua 1, Richard F Keep 1, Neeraj Chaudhary 1,3, Guohua Xi 1
PMCID: PMC5871578  NIHMSID: NIHMS943383  PMID: 29511126

Abstract

Background and Purpose

Brain iron overload is a key factor causing brain injury after intracerebral hemorrhage (ICH). This study quantified brain iron levels after ICH with magnetic resonance imaging (MRI) R2* mapping. The effect of minocycline on iron overload and ICH-induced brain injury in aged rats was also determined.

Methods

Aged (18-month-old) male Fischer 344 rats had an intracerebral injection of autologous blood or saline, and brain iron levels were measured by MRI R2* mapping. Some ICH rats were treated with minocycline or vehicle. The rats were euthanized at day 7 and 28 after ICH and brains used for immunohistochemistry and Western blot analyses. MRI (T2-weighted, T2* gradient-echo and R2* mapping) sequences were performed at different time points.

Results

ICH-induced brain iron overload in the perihematomal area could be quantified by R2* mapping. Minocycline treatment reduced brain iron accumulation, T2* lesion volume, iron handling protein upregulation, neuronal cell death, and neurologic deficits (p < 0.05).

Conclusions

MRI R2* mapping is a reliable and noninvasive method, which can quantitatively measure brain iron levels after ICH. Minocycline reduced ICH-related perihematomal iron accumulation and brain injury in aged rats.

Keywords: cerebral hemorrhage, iron, minocycline, MRI

Introduction

Brain iron overload has a significant role in brain injury after intracerebral hemorrhage (ICH)1. Iron accumulation in the brain after ICH causes edema, neuronal death and neurological deficits 2, 3. We have shown an increase in brain nonheme iron after ICH in rats that persists for at least several weeks 4 and deferoxamine, an iron chelator, can reduce ICH-induced brain damage 5.

It is important to develop reliable and noninvasive methods to quantify brain iron levels for experimental and clinical studies. Magnetic resonance imaging (MRI) can detect parenchymal hematomas and iron with high sensitivity. Tissue iron shortens T2* relaxation times measured by MRI. T2* gradient-echo imaging has been used to assess hematoma size early after ICH and brain iron accumulation at later phases 6, 7. The reciprocal of T2*, known as (relaxivity) R2*, is directly proportional to iron concentration with studies describing a near linear rise in R2* with iron concentration8, 9. Mapping of the relaxation rate R2* represents an indirect measure of iron 8, 10, 11.

Minocycline, a second-generation tetracycline-based molecule, is a potent inhibitor of microglia activation that can penetrate the brain-blood barrier easily. We and others have demonstrated that minocycline is able to chelate iron in vitro and in vivo 12, 13.

This study investigated the time course of perihematomal iron in an ICH model in aged rats using MRI R2* mapping. It then examined whether minocycline can reduce ICH-induced iron overload and brain injury.

Materials and Methods

The authors adhere to the Transparency and Openness Promotion (TOP) guidelines of the American Heart Association and declare that all supporting data are available within the article.

Animal preparation and intracerebral blood injection

The University Committee on Use and Care of Animals, University of Michigan approved the animal procedure protocols. The study complies with the ARRIVE guidelines for reporting in vivo experiments. A total of 67 18-month-old male Fischer 344 rats (National Institutes of Health, Bethesda, MD, USA) were used in this study. The ICH model was performed as previously described. Rats were anesthetized with pentobarbital (45 mg/kg intraperitoneally). Core body temperature was maintained at 37.5 ± 0.5 °C by a feedback-controlled heating pad. The right femoral artery was inserted with a polyethylene catheter to obtain blood for injection and to monitor arterial blood pressure, blood gases, and glucose concentrations. Rats were positioned in a stereotactic frame (Kopf Instruments, Tujunga, CA, USA), and a cranial burr hole (1.0 mm) was drilled on the right coronal suture 3.5mm lateral to midline. A 26-gauge needle was inserted stereotactically into the right basal ganglia (coordinates: 0.2mm anterior, 5.5 mm ventral, 3.5mm lateral to bregma). 100-µL autologous arterial blood or saline was injected over 10 min with a micro infusion pump.

Experimental groups

Rats had an intracaudate injection of 100-µL autologous arterial blood (ICH) or saline (control). The animals were randomized using selection of odd or even numbers. In the first part of study, rats were euthanized at day 7 or 28 after ICH or saline injection (n = 7–8 each group). Serial MRI scan was performed at different time points (day 1 to day 28). . In the second part of study, ICH rats were treated with minocycline (20 mg/kg, intraperitoneally, at 2 and 12 hours after ICH followed by 10 mg/kg twice a day up to 7 days) or vehicle (saline) (n = 8 each group). The minocycline dose was determined in our preliminary study. We found that high doses of minocycline (45 mg/kg, i.p., at 2 and 12 hours after ICH followed by 22.5 mg/kg twice a day up to 7 days) resulted in high mortality rate (approximately 50%) in aged rats. MRI scan was performed, and the rats were euthanized at day 7 or 28. All rats had behavioral tests. Harvested brains were used for immunohistochemistry and Western blot analysis.

Magnetic Resonance Imaging

Rats were anesthetized with 1.5~2% isoflurane throughout MRI examination. T2-weighted, T2* gradient-echo and T2* array imaging were performed in a 7.0-T Varian MR scanner (Varian, Palo Alto, CA, USA). T2-weighted and T2* gradient-echo imaging (repetition time/echo time = 4,000 ms/60 ms and 200 ms/5 ms for T2 and T2* imaging, respectively) were performed as described previously14. The T2 and T2* images were taken to measure ventricular and T2* lesion volumes. The bilateral ventricles and T2* lesion were outlined for area measurement in NIH image J. Total volumes were obtained by combining the areas and multiplying by the thickness (0.5 mm) of the sections. Ipsilateral ventricular volume was expressed as a percentage of contralateral.

T2* array imaging (repetition time = 250 ms, echo time = 6, 11, 16, 21, 26, 31, 36, 41 ms) was performed with a field of view of 35×35 mm and 5 coronal slices per echo time sequence (1.0 mm thick). The series images of T2* array were reconstructed at different echo times for analysis as R2* (1/T2*) mapping with Matlab software. Three areas in the perihematomal region (Fig 1A) and three corresponding areas in the contralateral hemisphere (Fig 1A) were measured. The perihematomal regions were determined on T2* images for each animal. A series of standard iron concentration (14, 28, 56, 112, 224 µg/ml; Ferric Chloride solution, 157740-5G, Sigma-Aldrich) solutions in test tubes were also scanned under T2* array. There was a near linear correlation between the R2* value on MRI images and the iron concentration of the standard solutions (Fig 1A). The brain iron content was calculated from that correlation.

Figure 1.

Figure 1

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(A) Left panel: Representative brain iron content measurement area on MRI T2*image on day 7 (3 squares in the ipsilateral hemisphere and 3 squares in the contralateral hemisphere). Right panel: The linear correlation between R2* value and iron content. (B) Time course of brain iron content after ICH in aged rats. Values are mean ± SD, n=8, *p < 0.05 vs. the contralateral side, **p < 0.05 vs. the ipsilateral side on day 1, two-way ANOVA.

Immunohistochemistry

Immunohistochemistry staining was performed as described previously14. Briefly, rats were anesthetized with pentobarbital (100 mg/kg, intraperitoneally) and perfused with 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (pH 7.4). Brains were harvested and kept in 4% paraformaldehyde for 24 hours, then immersed in 30% sucrose for 3 to 4 days at 4°C. After embedding in a mixture of 30% sucrose and optimal cutting temperature compound (Sakura Finetek, Inc., Torrance, CA, USA) at a ratio of 1:2, brains were sectioned at 18 µm on a cryostat. Immunohistochemistry studies were performed with avidin-biotin complex techniques. Primary antibodies were polyclonal rabbit anti-heme oxygenase-1 (HO-1, Enzo, ADI-SPA-895-F, 1:400 dilution), polyclonal rabbit anti-ferritin IgG (AbD; 1:500 dilution) and polyclonal rabbit anti-dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) Ig G (Cell Signaling, 1:500 dilution).

Enhanced Perls’ Reaction

Brain sections were incubated in Perls’ solution (1:1, 5% potassium ferrocyanide and 5% HCl) for 45 min, washed in distilled water, and incubated again in 0.5% diamine benzidine tetrahydrochloride with nickel for 60 min.

Western blots

Western blot analysis was performed as previously described. 14 Primary antibodies were polyclonal rabbit anti-HO-1 (Enzo, ADI-SPA-895-F, 1:1000 dilution), polyclonal goat anti-ferritin-L-chain IgG (FTL, Abnova, Walnut, CA, USA, 1:2000 dilution), polyclonal rabbit anti-ferritin-H-chain IgG (FTH, Cell Signaling, Beverly, MA, USA, 1:2000 dilution), polyclonal rabbit anti-DARPP-32 (Cell Signaling, Beverly, MA, USA, 1:10,000 dilution), monoclonal rabbit anti-β-actin (Cell Signaling, Beverly, MA, USA, 1:10,000 dilution) and monoclonal mouse anti-GAPDH (Fitzgerald, 10R-G109A, 1:10,000 dilution).

Behavioral Tests

Functional outcomes were assessed using forelimb-placing and corner turn tests15. The two behavioral tests were carried out by an investigator (YH) who was blinded to the treatment.

Statistical analysis

All the data are expressed as means ± SD. Data were analyzed by Student t tests or one- and two-way analysis of variance (ANOVA) with a Scheffe’s multiple comparisons test. Differences were considered significant at P < 0.05. A power analysis indicated that an n=8 in the vehicle and minocycline groups would have greater than 80% power to detect a 25% change in T2* lesion volume. The same group sizes were estimated to have 95% power to detect a 15% change in tissue iron.

Results

Mortality rate after ICH was low in this aged rat study. A total of 4 rats died during the experiments, including two in the first set of experiments (two ICH rats), two in the second set (one vehicle-treated and one minocycline-treated rat). Significant side effects resulting from minocycline treatment, such as diarrhea, were not observed during our experiments.

Intracerebral hemorrhage induces iron deposition in perihematomal brain tissue

MRI R2* mapping was used to determine brain iron content in perihematomal area. Iron levels in the hematoma were too high for R2* mapping. The near linear correlation between R2* value and iron content was excellent from 14 to 224 µg/ml in test tubes (correlation coefficient = 0.9996; Fig 1A). Using the contralateral hemisphere as a control, we found that the brain iron content around the hematoma increased gradually over the time after ICH with a peak at day 14, and stayed high at one month (Fig 1B). Compared to controls receiving an intracerebral injection of saline, brain iron content was significantly increased in the side ipsilateral to the hematoma but not the contralateral hemisphere at day 7 after ICH (Fig 2).

Figure 2.

Figure 2

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Brain iron content in the ipsilateral and contralateral basal ganglia at day 7 after ICH or saline control. Values are mean ± SD, n=7–8, #p < 0.01 vs. the ipsilateral basal ganglia in the saline group, Student t test.

Minocycline reduces brain iron overload after ICH in aged rats

Whether minocycline can attenuate brain iron overload after ICH in aged rats was examined. Minocycline reduced brain iron levels in perihematomal area at both day 7 (73.4 ± 8.4 versus 88.0 ± 9.7 µg/ml in the vehicle-treated ICH animals, p < 0.01) and day 28 (76.4 ± 9.4 versus 92.9 ± 10.4 µg/ml with vehicle treatment, p < 0.01). By enhanced Perls’ staining, an iron-positive area was also found in the perihematomal zone, corresponding to the images from MRI R2* mapping (Fig 3A). Our previous study demonstrated that MRI T2* changes in the brain correlated closely with the hematoma in the acute phase and brain iron overload in the chronic phase following ICH6. Thus, the volume of T2* was determined. The T2* lesion volume was decreased in the minocycline-treated group both at day 7 (33.6 ± 7.8 versus 39.9 ± 10.0 mm3 in the vehicle-treated group, p < 0.05, Fig 3B) and day 28 (21.7 ± 3.7 versus 33.5 ± 4.3 mm3 with vehicle treatment, p < 0.01, Fig 3B).

Figure 3.

Figure 3

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(A) Representative brain sections, Perls’ staining, MRI R2* and T2* images of vehicle- and minocycline-treated groups at days 7 and 28 after ICH. (B) MRI T2* lesion volumes in vehicle- and minocycline-treated groups at days 1, 7 and 28 after ICH. Values are mean ± SD, n=8, *p < 0.05, #p < 0.01 vs. the vehicle-treated group, Student t test.

HO-1 is a key enzyme for heme degradation. HO-1 was upregulated in the perihematomal zone after ICH in aged rats, and minocycline attenuated the increased HO-1 expression compared with vehicle treatment at day 7 (p < 0.05; Fig 4A). Ferritin, an iron storage protein, was associated with brain iron deposition after ICH. Ferritin-positive cells were less in minocycline-treated rats at day 7 (Fig 4B). Western blot analysis also showed that both ferritin-L- and H-chain levels were lower in the minocycline-treated group compared with the vehicle-treated group at day 7 (p < 0.05; Fig 4B).

Figure 4.

Figure 4

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(A) HO-1 immunoreactivity and protein levels in saline control, vehicle- and minocycline-treated ICH animals at day 7. Scale bar = 50 µm. Values are mean ± SD, n=4, #p < 0.01 vs. saline group, *p < 0.05 vs. minocycline-treated group. (B) Ferritin immunoreactivity and protein levels of ferritin-L-chain and ferritin-H-chain in saline control, vehicle- and minocycline-treated ICH animals at day 7. Scale bar = 50 µm. Values are mean ± SD, n=4, #p < 0.01 vs. saline control group, *p < 0.05 vs. minocycline-treated ICH animals, #p < 0.01 vs. the other groups, one-way ANOVA.

Minocycline attenuates ICH-induced neuronal death, neurological deficits and brain atrophy in aged rats

DARPP-32, a specific marker of neurons in the basal ganglia, was used as a simple and reliable maker for neuronal injury after experimental ICH. Using Western blot analysis and immunochemistry staining, DARPP-32 levels in the ipsilateral basal ganglia were decreased significantly after ICH compared with saline group (p < 0.05; Fig 5) at day 7. Minocycline treatment reduced this ICH-induced caudate neuronal death (p < 0.05; Fig 5).

Figure 5.

Figure 5

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Immunoreactivity (A) and protein levels (B) of DARPP-32 in saline control, vehicle- and minocycline-treated ICH animals at day 7. Scale bar = 50 µm. Values are mean ± SD, n=4, *p < 0.05 vs. the other groups, one-way ANOVA.

To assess behavioral deficits, forelimb placing and corner turn test were used. Compared to controls receiving an intracerebral saline injection, ICH caused significant neurological deficits at day 1 in both minocycline-treated and vehicle-treated groups. At days 7 and 28, however, the minocycline-treated ICH group had less neurological deficits than vehicle-treated rats (p < 0.05; Fig 6).

Figure 6.

Figure 6

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Forelimb placing (A) and corner turn (B) scores in saline control, vehicle- and minocycline-treated ICH animals at day 1, 7 and 28 after ICH. Values are mean ± SD, n=7–15, *p < 0.05 vs. vehicle-treated groups, #p <0.01 vs. the other groups, one-way ANOVA.

To estimate brain atrophy, we measured ventricular enlargement at day 28 after ICH. T2-weighted MRI showed marked enlargement of the ipsilateral ventricle after ICH. That enlargement was significantly less with minocycline treatment (189 ± 97%) compared to vehicle-treatment (314 ± 92%, n=8; p < 0.05).

Discussion

The major findings of this study are as follows: (1) MRI R2* mapping was a reliable and noninvasive method of quantitative measure for brain iron overload after ICH; (2) minocycline treatment reduced brain iron overload after ICH in aged rats; and (3) minocycline attenuated ICH-induced brain injury, and improved functional outcome in aged rats.

Iron, a hemoglobin degradation product, has a major role in brain damage following ICH1. Iron can accumulate in the perihematomal zone after erythrocyte lysis following ICH, contributing to acute brain edema formation and delayed brain atrophy 2, 3, 16. Both early brain edema and delayed brain tissue loss cause neurological deficits after ICH. Iron content in the perihematomal area could be high for several weeks after ICH. Our previous biochemical study showed that there was a 3-fold increase of brain nonheme iron in the perihematomal zone after ICH in young rats 4. ICH is, however, mostly a disease of the elderly and this study using MRI R2* mapping detected the natural time course of brain iron accumulation in perihematomal zone in the aged rat ICH model. It should be noted that the pattern of brain iron overload measured by MRI was similar to that determined by non-heme iron assay 4.

Although there is a large body of evidence for the role of iron in brain damage following parenchymal hemorrhage, there is lack of reliable and noninvasive methods for accurate quantification of the tissue iron content. Following such demands, existing noninvasive modalities such as MRI have been utilized to develop iron measurement not only in the brain tissue, but also in the liver and heart8–10. Paramagnetic effects of tissue-deposited iron, as a form of signal inhomogeneity, have been exploited to estimate tissue iron content. Our previous study demonstrated that T2*-weighted MRI changes in the brain correlate closely with brain iron overload following ICH6. Another study showed the feasibility of brain iron measurement in a normal aging population by quantitative susceptibility mapping17. With the further studies in this field, many investigators described the near linear rise in R2*(1/T2*) and R2 (1/T2) signal with iron content8, 9, 18, 19. Mapping of the relaxation rate R2* represents an indirect measure of iron. It has been reported that MRI R2* mapping could accurately estimate hepatic iron content in sickle cell disease patients8. Our group has demonstrated proof of principle for brain iron measurement by using MRI R2* mapping in two ICH patients 20. The present study was to quantitatively detect perihematomal brain iron content by MRI R2* mapping in the aged rat ICH model. The results suggested a gradual increase of iron accumulation in perihematomal zone in aged rat, and a plateau around 2 weeks after ICH. The MRI brain tissue iron quantification may provide a non-invasive way for examining brain iron levels after hemorrhagic stroke in animals and in patients. However, it was not possible to measure iron levels in the hematoma at acute phase (too high) or in the cavity at chronic phase after ICH.

Minocycline has been reported to provide neuroprotection in both hemorrhagic and ischemic animal models12, 21, 22. Our previous studies have demonstrated that minocycline acts as an iron chelator and an inhibitor of microglial activation, and can reduce ICH-induced brain iron overload and brain injury21. Another in vitro study showed that minocycline could attenuate iron neurotoxicity in cortical neuronal cultures23. Further research has proven that the iron chelate effect of minocycline, but not the inhibition effect of microglial activation, attenuated iron-induced brain edema and BBB disruption12. In the current study, we demonstrated that minocycline reduced ICH-induced brain injury in aged rats with decreasing iron deposition in perihematomal zone. We hypothesize that minocycline chelates iron within or close to the hematoma preventing the spread of iron into perihematomal tissue. As an antibiotic, minocycline can be administered orally, and it maintains effective blood drug concentration through intestinal absorption. It is also a highly lipophilic compound that penetrates the blood-brain barrier easily. The results suggested that minocycline may be a promising treatment for ICH patients.

The mechanisms of minocycline-induced brain protection after ICH are complicated. It is well known that minocycline is an inhibitor of microglia, but it is also an iron chelator12. In the current study, we found that minocycline reduces perihematomal HO-1 expression at day 7 and the most of HO-1 positive cells are microglia/macrophages. It is still unclear whether reduced HO-1 expression in minocycline-treated animals results from microglia inhibition, iron chelation or both. Microglia/macrophage polarization has an important role in hematoma clearance and brain recovery following ICH24, 25, and future studies should examine the role of minocycline in microglia/macrophage polarization, clot resolution and brain recovery. It should be noted that minocycline inhibited M1 polarization in a mouse model of amyotrophic lateral sclerosis.26

Minocycline also reduces ICH-induced upregulation of ferritin in the perihematomal zone. Most of ferritin positive cells are glia. Our previous study demonstrated that perihematomal ferritin positive cells are microglia and astrocytes.4 Because astrocytes have a role in brain iron overload27, it will be interesting to examine the effects of minocycline on astrocyte activation following ICH.

The current study focused on brain iron quantification by MRI R2* mapping and the effect of minocycline on ICH-induced brain injury in aged rats. There are several limitations in this study: (1) it is unclear whether MRI R2* mapping measures intracellular or extracellular iron, whether R2* signal may change during changes in iron oxidation or protein binding; (2) the correlation between iron standards and R2* was determined in test tubes; (3) minocycline was only tested in aged male rats. It is important to examine whether minocycline also can reduce ICH-induced brain iron overload and brain injury in aged female animals; (4) only one dose of minocycline was tested. Further translational studies should determine optimal dose, optimal durations and therapeutic window for minocycline.

In conclusion, brain iron accumulation occurs after ICH in aged rats, which can be measured by MRI R2* mapping. Minocycline reduces ICH-induced brain iron overload as assessed by R2* mapping and brain injury in aged rats, and could be an effective treatment for ICH patients. MRI R2* mapping is a method for quantitatively assessing iron overload and iron-focused treatments in cerebral hemorrhage.

Supplementary Material

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Visual Abstract

Acknowledgments

Sources of Funding: This study was supported by grants NS-091545, NS-090925, NS-096917 and NS-099684 from the National Institutes of Health (NIH), 973 Program-2014CB541600, a University of Michigan/Peking University Joint Institute grant and the Joyce & Don Massey Family Foundation.

Footnotes

Conflict of Interests: the authors declared no conflict of interests.

References

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Associated Data

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Supplementary Materials

Modified_Checklist
NIHMS943383-supplement-Modified_Checklist.pdf (76.3KB, pdf)
Visual Abstract
NIHMS943383-supplement-Visual_Abstract.pdf (1.2MB, pdf)

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