Background and Purpose: We have previously demonstrated that 2-week treatment of experimental intracerebral hemorrhage (ICH) with a daily dose of 2 mg/kg statin starting 24 hours post-injury exerts a neuroprotective effect. The present study extends our previous investigation and tests the effect of acute high-dose (within 24 hours) statin therapy on experimental ICH. Material and Methods: Fifty-six male wistar rats were subjected to ICHby stereotactic injection of 100 μl of autologous blood into the striatum. Rats were divided randomly into seven groups: saline control group (n = 8); 10, 20 and 40 mg/kg simvastatin-treated groups (n = 8); and 10, 20 and 40 mg/kg atorvastatin-treated groups (n = 8). Simvastatin or atorvastatin were administered orally at 3 and 24 hours after ICH. Neurological functional outcome was evaluated using behavioral tests (mNSS and corner turn test) at multiple time points afterICH. Animals were sacrificed at 28 days after treatment, and histological studies were completed. Results: Acute treatment with simvastatin or atorvastatin at doses of 10 and 20 mg/kg, but not at 40 mg/kg, significantly enhanced recovery of neurological function starting from 2 weeks post-ICH and persisting for up to 4 weeks postICH. In addition, at doses of 10 mg/kg and 20 mg/kg, histological evaluations revealed that simvastatin or atorvastatin reduced tissue loss, increased cell proliferation in the subventricular zone and enhanced vascular density and synaptogenesis in the hematoma boundary zone when compared to salinetreated rats. Conclusions: Treatment with simvastatin or atorvastatin at doses of 10 and 20 mg/kg significantly improves neurological recovery after administration during the first 24 hours after ICH. Decreased tissue loss, increased cell proliferation and vascularity likely contribute to improved functional recovery in rats treated with statins after ICH.
Intracerebral hemorrhage (ICH) is a devastating form of stroke subtype with high rates of mortality and morbidity [1,2]. Current clinical treatment strategies for ICH involve a combination of medical and surgical intervenetions to reduce bleeding and control hypertension and elevated intracranial pressure (ICP). Current standards of care are not sufficient to improve the clinical outcomes of patients who suffer from ICH [
Statins, which are HMG-CoA reductase inhibitors, are widely prescribed as cholesterol-lowering medications and have also been shown to exert beneficial pleiotropic effects on neurological diseases [4,9-11]. These effects include reduction of neuroinflammation and oxidative stress, improvement of vascular endothelial function, and enhancement of synaptogenesis [11-14]. Emerging evidence, however, has revealed that high-dose statins may have a negative impact on angiogenesis, an important tissue repair mechanism in the neurodegenerative diseases [15,16]. Moreover, in patients with a prior history of stroke, aggressive reduction in cholesterol levels with high-dose atorvastatin (SPARCL trial) increased the incidence of ICH [
All experimental procedures were approved by the Institutional Animal Care and Use Committee at Henry Ford Health System (IACUC #0814). Adult male Wistar rats (n = 56, weighing 270 - 330 g) were subjected to ICH by stereotactic infusion of 100 µl of autologous whole blood into the striatum region adjacent to the subventricular zone (SVZ) [4,18]. After the ICH, animals were randomly divided into seven groups: 1) saline control group (n = 8); 2) 10 mg/kg simvastatin-treated group (n = 8); 3) 10 mg/kg atorvastatin-treated group (n = 8); 4) 20 mg/kg simvastatin-treated group (n = 8); 5) 20 mg/kg atorvastatin-treated group (n = 8); 6) 40 mg/kg simvastatintreated group (n = 8); and 7) 40 mg/kg atorvastatintreated group (n = 8). Simvastatin or atorvastatin or saline was administered orally at 3 and 24 hours [
Functional outcome was assessed using a corner turn test [
At the end of 4 weeks, the animals were anesthetized and perfused transcardially with PBS, followed by 4% paraformaldehyde in PBS. Brain tissues were excised, further fixed in 4% paraformaldehyde and sliced into seven 2- mm-thick sections. Each block was processed and embedded in paraffin. Four of the adjacent 6-mm-thick sections were cut from each block and were stained with hematoxylin and eosin (H & E).
The brain tissue residing between +0.1 and 0.86 mm of the bregma on the third block was the most severely injured and therefore the third block was specifically selected for immunostaining. Every 40th coronal section from +0.1 and 0.86 mm of the bregma was used for immunohistochemical staining with the same antibody. Sections were blocked in a Tris buffered saline solution containing 5% normal goat serum, 1% BSA and 0.05% Tween-20. Sections were then incubated with the primary antibodies for localization of BrdU (1:100; a marker for proliferation cells), doublecortin (DCX) (1:50; a microtubule-associated protein expressed almost exclusively in immature neurons), synaptophysin (1:1000; a marker for a protein located on synaptic vesicles which contain the neurotransmitters for the labeling of nerve terminals) and EBA (endothelial barrier antigen) (1:1000, a marker for detection of mature vessels). For DAB staining, the sections were incubated with biotinylated IgG and followed with an avidin-biotin-peroxidase system (ABC Kit, Vector Laboratories, Burlingame, CA). DAB was then used as a sensitive chromogen for light microscopy. For immunofluorescent staining, the sections were incubated with Cy3- and/or FITC-conjugated antibody (1:200; Jackson ImmunoResearch) at room temperature for 2 hours.
After staining with BrdU, DCX, synaptophysin, and EBA, a series of six slides at various levels from the same block were used for semiquantitative measurements. Quantitative measurements of immunostaining were performed by an observer blinded to the individual treatment status of the animals. All slides were digitized under a ×20 objective lens (Nikon, Eclipse 80i, Melville, NY) using a CoolSNAP color camera (Photometrics, Tucson, AZ) interfaced with a MetaMorph image analysis system (Molecular Devices, Downingtown, PA). BrdU+ cells and DCX+ signals in the dorsal end of the lateral ventricular zone with 100-mm width and 800-mm length were digitized. The synaptophysin immunoreactive area in the perihemorrhagic striatum was measured. Data are presented as percentage of synaptophysin immunoreactive areas in each field divided by the total area in the field. For quantification of vessels, the number of EBA positive vessels was measured in eight fields of view within the hematoma boundary zone area. Data are presented as the density of EBA immunoreactive vessels relative to the area of the hematoma boundary zone.
Statistical analysis of neurological functional scores, areas of ICH-related tissue loss, and immunohistochemical results were obtained using ANOVA. Data are presented as the mean ± SEM, and probability value < 0.05 were considered significant. All measurements were performed by observers blinded to individual treatments.
As evaluated by both mNSS and corner turn tests, all ICH-induced rats had similar neurological impairments prior to and at one week after ICH (
The data for the percentage of striatal tissue loss on the side of hemorrhage is shown in
To determine the effects of statins on SVZ cell proliferation, immunostaining for BrdU was performed (
To investigate the effects of simvastatin and atorvastatin on neurorestoration, histology and immunohistochemistry were performed. Typical immunostainings for synaptophysin in the control group and the treatment group are presented in
To evaluate the effect of statin therapy on vessel proliferation and density, quantitative measurements of EBA staining were performed. When compared to controls, rats treated with 10 and 20 mg/kg had significantly increased density (vessels/mm2) of EBA-positive vessels in the hematoma boundary zone area (p < 0.05) compared with saline-treated rats (
This study demonstrates a dose-dependent effect of highdose statins administered acutely after experimental ICH. Simvastatin and atorvastatin at doses of 10 and 20 mg/kg significantly improved neurological function at 4 weeks post-ICH, whereas the higher dose of 40 mg/kg did not significantly improve neurological function. These findings are consistent with pathologic findings of signifycantly less tissue loss for the lower dose and more tissue loss for the higher dose. The therapeutic benefits observed with statins at 10- and 20-mg/kg doses may partially be attributed to increased cell proliferation, vascularity and synaptogenesis, as demonstrated by histological analysis.
Currently, patients are admitted to the hospital early (within 3 hours) after ICH onset because of the profound ictus. However, Valiente et al. found a higher 30-day
mortality and a worse outcome for ICH patients admitted within the 3-hour post ictus [
In our previous study, when atorvastatin was administered for 2 weeks with a low dose of 2 mg/kg, a significant reduction in neurological deficit two to four weeks after the ICH was observed, while a higher dose of 8 mg/kg did not improve functional outcome or reduce brain damage [
Several potential mechanisms such as antioxidative, anti-inflammatory, anti-apoptosis, neurotrophism and angiogenesis may contribute to the neuroprotective and neurorestorative effects of statins in experimental ICH [4,11,12,29]. Our data demonstrated that acute treatment with simvastatin and atorvastatin has the ability to reduce tissue loss, improve neurorestoration and improve functional recovery after ICH in a dose-limited manner. An interesting finding is that treatment with a statin dose of 20 mg/kg trended towards being slightly more effective at reducing tissue loss compared with a treatment dose of 10 mg/kg. However, the values for neurorestorative markers demonstrated a reverse relationship. This finding suggests that the improved neurological outcome with statin treatment after ICH may be attributed to several potential mechanisms, which individually may have different dose sensitivities, but together promote functional recovery.
There are limitations in the present study. We hypothesized that the effects of low cholesterol in association with the higher dose of statins might be linked to a reduction in functional recovery, but we did not measure plasma cholesterol level. Sironi et al. have demonstrated that simvastatin pretreatment (20 mg/kg for 3 days before MCAO) or post-treatment (20 mg/kg for 2 days after MCAO) did not affect the plasma cholesterol levels [
In conclusion, statin treatment at 3- and 24-hours post ICH is associated with neuronal and functional recovery after experimental ICH. Acute statin therapy 3 and 24 hours after ICH may have significant clinical implications, since most ICH patients would be under medical care at these early time points.