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This study tested the hypothesis that extracorporeal shock wave (ECSW) therapy could protect mouse brain from chronic cerebral hypoperfusion (CHP)-induced neuropathological changes in a bilateral carotid arterial stenosis (CAS) model. Adult-male C57BL/6 (B6) mice (n=36) were randomized into group 1 (sham-control), group 2 (CHP) and group 3 [CHP+ECSW (100 impulses at 0.15 mJ/mm2) on day 5, 10 and 15 after CHP induction]. By day 60 after CHP induction, the white matter lesion, protein expressions of inflammatory (TNF-α/NF-κB/iNOS), oxidative-stress (NOX-1/NOX-2/NOX-4/nitrotyrosine), angiogenesis (eNOS/CD31), apoptotic (Bax/caspase-3/PARP), fibrotic (Smad3/TGF-ß) and mitochondrial-damaged (cytosolic cytochrome-C) biomarkers were significantly higher in group 2 than in groups 1 and 3, and significantly higher in group 3 than in group 1, whereas the protein expressions of anti-apoptotic (Bcl-2), anti-fibrotic (BMP-2/Smad1/5), and mitochondrial-integrity (mitochondrial cytochrome-C) biomarkers showed an opposite pattern to inflammation among the three groups (all P<0.0001). The cellular expressions of inflammatory (Iba-1/GFAP/CD14, F4/80), apoptotic (TUNEL-assay) and brain-damaged (γ-H2AX/AQP4) biomarkers showed an identical pattern to inflammation, whereas the cellular expressions of endothelial-cell (CD31/vWF), neuron/energy-integrity (NeuN/PGC-1α) and small-vessel density exhibited an opposite pattern to inflammation among the three groups (all P<0.0001). Cellular angiogenesis (VEGF/SDF-1α) significantly and progressively increased from groups 1 to 3 (all P<0.0001). In conclusion, ECSW therapy enhanced angiogenesis, inhibited molecular-cellular perturbations, and protected the white matter and neuron from CHP damage.
Dementia represents a syndrome of acquired intellectual deficits resulting in significant impairment of social and occupational function and is increasingly an economic burden worldwide [1-4]. The possible etiologies for dementia are diverse , but ischemic cerebrovascular disease (CVD) is considered a major cause especially in the elderly. The spectrum of vascular cognitive impairments caused by various types of CVD occur due to interactions between a variety of vascular risk factors such as hypertension, obesity, dyslipidemia, diabetes mellitus, chronic ischemic stroke (IS) and silent IS [6-8].
Age-related chronic cerebral hypoperfusion (CHP) is commonly caused by carotid artery stenosis and atherosclerotic arterial occlusive diseases of larger and small intracranial vessels, which result from various etiologies that lead, subsequently, to ischemic white matter lesions (WML) [6,8] and later dementia. Despite pharmacological advances and carotid artery stenosis interventions, an effective treatment for CHP-related dementia is still lacking [1,5,8].
Compared with other organs, the brain is more susceptible to damage from oxidative-stress due to its high metabolic rate, high polyunsaturated lipid content, and lower levels of endogenous antioxidant activity and self-protection mechanisms. WML caused by chronic CHP may play an essential role in the development of vascular-impaired dementia (VID) via the upregulation of oxidative stress and inflammation [9-14]. Thus, a therapeutic modality for dementia in chronic CHP patients could involve abrogating oxidative stress and inflammation as well as restoration of blood flow through enhancing angiogenesis in the hypoperfused brain.
Extracorporeal shock wave (ECSW) therapy is well known for providing a mechanical means of treatment, such as lithotripsy for kidney and urethral stones. Its low-energy form (ranging from 0.03 to 0.15 mJ/mm2) can also elicit a series of subtle biological effects for treating the musculoskeletal [15,16] and cardiovascular systems [17-20]. Studies have also shown that ECSW therapy improves ischemia-related organ dysfunction by suppressing the inflammatory response and the generation of reactive oxygen species (ROS), and by enhancing angiogenesis/neovascularization [17-21]. However, whether ECSW can effectively reverse CHP-induced WM lesions and VID has not been investigated. Accordingly, the present study used a mouse chronic CHP model (i.e., induction of bilateral carotid arterial stenosis using micro-coils) to test the hypothesis that ECSW therapy could effectively inhibit chronic CHP-induced brain damage.
All animal experimental protocols and procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2015062302) and performed in accordance with the Guide for the Care and Use of Laboratory Animals [The Eighth Edition of the Guide for the Care and Use of Laboratory Animals (NRC 2011)].
Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-approved animal facility in our hospital (IACUC protocol no. 101008) with controlled temperature and light cycles (24°C and 12/12 light cycles).
The experimental protocol was based on our previous report with minimal modification . Twelve-week old male C57Bl/6J mice (n=36) weighing 25-30 g (Charles River Technology, BioLASCO Taiwan Co. Ltd., Taiwan) were categorized into sham control (SC, n=12), CHP (induced by bilateral carotid artery stenosis procedure, n=12], and CHP + ECSW [(100 impulses at 0.15 mJ/mm2) on day 5, 10 and 15 after CHP induction, n=12].
The protocol and procedure were based on our previous report . All animals were anesthetized (inhalational 2.0% isoflurane) and placed supine on a warming pad at 37°C during the BCAS procedure. Both sides of the neck were incised and the carotid arteries identified. Bilateral carotid artery stenoses were induced by encasing both common carotid arteries within micro-coils (Sawane Spring Co, Shizuoka, Japan; internal diameter 0.18 mm) to cause luminal narrowing. Sham-control mice received an identical operation without micro-coil placement on the carotid arteries.
The procedure and protocol for specimen collection have previously been reported [4,19,22-24]. For examination of protein expression, animals in all groups were sacrificed on day 60 after brain acute IS procedure, and the brain of each rat was promptly removed, immersed in cold saline, snap-frozen in liquid nitrogen and then stored at -80°C for individual study. For immunofluorescent (IF) and immunohistochemical (IHC) staining studies, the brains of 6 other animals in each group were reperfused with normal saline via the carotid artery, removed, fixed with 4% paraformaldehyde in 1× PBS (pH 7.4), and soaked in 20% sucrose in 1× PBS (freshly prepared) until the brain took on a completely sunken appearance. The sucrose was then discarded and the brain soaked in 30% sucrose in 1× PBS (freshly prepared) for 48 h; the tissue specimen was then retrieved. Finally, an OCT block (Tissue-Tek, Sakura, Netherlands) was prepared for IHC and IF staining.
Kluver-Barrera staining was used to determine the WM lesions. We counted the number of vacuolation of neurons in internal capsule/corpus callosum (i.e., WM lesions) of cortex in 4 animals from each group.
The procedure and protocol of IF and IHC were based on our previous reports [4,19,22-24]. In detail, frozen sections (4 µm thick) were obtained from BIA of each animal, permeated with 0.5% Triton X-100, and incubated with antibodies against NeuN (1:1000, Millipore, Billerica, MA, USA), glial fibrillary acid protein (GFAP; 1:500, DAKO, Carpinteria, CA, USA), aquaporin4 (AQP4; 1:200, Abcam, Cambridge, MA, USA), Iba-1 (1:500; wako, Chuo-ku, Osaka, Japan), CD14 (1:200, Bioss, Massachusetts, Boston, USA), F4/80 (1:100; Abcam, Cambridge, MA, USA), γ-H2AX (1:1000; Abcam, Cambridge, MA, USA), vascular endothelial cell growth factor (VEGF) (1:400; Abcam, Cambridge, MA, USA), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) (1:100; Novus, Oakville, Ontario, Canada), stromal cell-derived factor (SDF)-1α (1:100, Santa Cruz Biotechnology), white matter lesion (WML) (i.e., Kluver-Barrera staining), TUNEL assay (In Situ Cell Death Detection Kit POD, Roche, Basel, Switzerland), α-SMA (1:400, Sigma-Aldrich, St. Louis, Missouri, USA), and von Willebrand Factor (vWF) (1:200, Millipore, Massachusetts, USA) at 4°C overnight. Alexa Fluor488, Alexa Fluor568, or Alexa Fluor594-conjugated goat anti-mouse or rabbit IgG were used to localize signals. Sections were finally counterstained with DAPI and observed with a fluorescent microscope equipped with epifluorescence (Olympus IX-40).
Three brain sections were analyzed for each mouse. For quantification, three randomly selected high-power fields (HPFs; ×400 for IHC and IF studies) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.
The procedure and protocol of Western blot were based on our previous reports [4,22-24]. Equal amounts (50 μg) of protein extracts were loaded and separated by SDS-PAGE using 12% acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Amersham, UK). Nonspecific sites were blocked by incubation of the membrane in blocking buffer [5% nonfat dry milk in T-TBS (TBS containing 0.05% Tween 20)] overnight. The membranes were incubated with monoclonal antibodies against NOX-1 (1:1500, Sigma-Aldrich), NOX-2 (1:1000, Sigma-Aldrich), NOX-4 (1:1000, Abcam), nitrotyrosin (1:1000, Abcam), tumor necrosis factor alpha (TNF-α) (1:1000, Cell Signaling), nuclear factor (NF)-κB (1:1000, Abcam), inducible nitric oxide synthase (iNOS) (1:200, Abcam), Cytochrome C (1:2000, BD Bioscience, San Jose, CA, USA), Aquaporin 4 (1:750, Abcam), nuclear factor (NF)-κB (1:1000, Abcam), endothelial nitric oxide synthase (eNOS) (1:1000, Abcam), CD31 (1:1000, Abcam), Bax (1:1000, Abcam), Caspase 3 (1:1000, Cell Signaling), Poly (ADP-ribose) polymerase (PARP) (1:1000, Cell Signaling), Bcl-2 (1:600, Abacm), cytosolic cytochrome C (1:1000, BD), mitochondrial cytochrome C (1:1000, BD), phosphorylated (p)-Smad3, p-Smad1/5, transforming growth factor (TGF)-ß (1:5000, Abcam), and bone morphogenetic protein (BMP)-2 (1:500, Abcam) for 1 hr at room temperature. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin IgG (1:2000, Cell Signaling) was used as a second antibody for 1 hr at room temperature. The washing procedure was repeated eight times within 1 h, and immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) and exposure to Medical X-ray film (FUJI). For quantification, ECL signals were digitized using Labwork software (UVP, Waltham, MA, USA). A standard control sample was loaded on each gel.
For each rat, six sections of BIA were analyzed with an in situ Cell Death Detection Kit, AP (Roche) according to the manufacturer’s guidelines. Three randomly chosen high-power fields (HPFs) (×400) were observed for terminal deoxynucleotidyltransferase-mediated 2’-deoxyuridine 5’-triphosphate nick-end labeling (TUNEL)-positive cells for each section. The mean number of apoptotic nuclei per HPF for each animal was obtained by dividing the total number of cells by 18.
The procedure and protocol for evaluating the protein expression of oxidative stress have previously been described [4,22-24]. The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon, Billerica, MA, USA (S7150). DNPH derivatization was carried out on 6 μg of protein for 15 minutes according to the manufacturer’s instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes that were then incubated in the primary antibody solution (anti-DNP 1:150) for 2 hours, followed by incubation in secondary antibody solution (1:300) for 1 hour at room temperature. The washing procedure was repeated eight times within 40 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Amersham, UK) and then exposed to Biomax L film (Kodak, Rochester, NY, USA). For quantification, ECL signals were digitized using Labwork software (UVP, Waltham, MA, USA). For oxyblot protein analysis, a standard control was loaded on each gel.
Quantitative data are expressed as mean ± SD. Statistical analysis was performed by ANOVA followed by Bonferroni multiple-comparison post hoc test. All analyses were conducted using SAS statistical software for Windows version 8.2 (SAS institute, Cary, NC). A probability value <0.05 was considered statistically significant.
The protein expressions of TNF-α, NF-κB, and iNOS, three indicators of inflammation, were significantly higher in CHP than in SC and CHP-ECSW, and significantly higher in CHP-ECSW than in SC. Protein expressions of eNOS and CD31, two indicators of endothelial-integrity, showed an opposite pattern to inflammation among the three groups.
The protein expression of cytosolic cytochrome C, an indicator of mitochondrial damage was significantly higher in CHP than in SC and CHP-ECSW, and significantly higher in CHP-ECSW than in SC. Protein expression of mitochondrial cytochrome C, an indicator of mitochondrial integrity, showed an opposite pattern to cytosolic cytochrome C among the three groups.
The protein expressions of NOX-1, NOX-2, NOX-4 and nitrotyrosine, four indicators of oxidative stress, were significantly higher in CHP than in SC and CPH-ECSW, significantly higher in CHP-ECSW than in SC and CHP-ECSW, and significantly higher in CHP-ECSW than in SC. Protein expression of VEGF, an indicator of angiogenesis, significantly increased from SC to CHP-ECSW, suggesting that an increase in VEGF was an intrinsic response to ischemic stimulation and further enhanced by ECSW therapy.
The protein expressions of mitochondrial Bax, cleaved caspase 3 and cleaved PARP, three indicators of apoptosis, were significantly higher in CHP than in SC and CHP-ECSW, and significantly higher in CHP-ECSW than in SC. Protein expression of Bcl-2, an indicator for anti-apoptosis, exhibited an opposite pattern to apoptosis among the three groups.
The protein expressions of Phospho-Smad3 and TGF-ß, two indicators of fibrosis, were significantly higher in CHP than in SC and CHP-ECSW, and significantly higher in CHP-ECSW than in SC, whereas, the protein expressions of Phospho-Smad1/5 and BMP-2, two indicators of anti-fibrosis, displayed an opposite pattern to fibrosis among the three groups.
The cellular expressions of Iba-1 and GFAP, two inflammation indicators in brain parenchyma, were significantly higher in CHP than in SC and CHP-ECSW, and significantly higher in CHP-ECSW than in SC.
Microscopy demonstrated that the cellular expressions of CD14 and F4/80, two cellular indicators of innate immunity, were significantly higher in CHP than in SC and CHP-ECSW, and significantly higher in CHP-ECSW than in SC.
IF microscopy showed that the number of AQP4+ cells, a biomarker for brain edema, was significantly higher in CHP than in SC and CHP-ECSW, and significantly higher in CHP-ECSW than in SC. Additionally, IF microscopy showed that the cellular expression of γ-H2AX, a biomarker for DNA-damage, exhibited an identical pattern to apoptotic nuclei.
TUNEL assay showed that the number of apoptotic nuclei was significantly higher in CHP than in SC and CHP-ECSW and significantly higher in CHP-ECSW than in SC. Additionally, the number of vacuole neurons in WM, an indicator for WML, displayed an identical pattern to apoptotic nuclei among the three groups. On the other hand, the number of normal architectural neurons (i.e., normal neurons) exhibited an opposite pattern of apoptosis among the three groups.
IF microscopy for cellular expressions of CD31 and vWF, two endothelial cell biomarkers, were significantly higher in SC than in CHP and CHP-ECSW, and significantly higher in CHP-ECSW than in HCP (Figure 8).
Microscopy for cellular expressions of VEGF and SDF-1α, two indicators of angiogenesis, were significantly increased from SC to CHP-ECSW (Figure 9).
IF microscopy showed that the cellular expression of NeuN, indicating neuron integrity, was significantly higher in SC than in CHP and CHP-ECSW, and significantly higher in CHP-ECSW than in CHP. Cellular expression of PGC-1α, an indicator of energy preservation, and number of small vessels, an indicator for angiogenesis/neovascularization, exhibited an identical pattern to neuN among the three groups.
IHC microscopy showed that the number of the small vessels, an indicator of angiogenesis/neovascularization in cerebral tissue, was significantly higher in SC than in CHP and CHP-ECSW, and significantly higher in CHP-ECSW than in CHP.
This study investigated the impact of ECSW treatment on protecting the brain against chronic CHP damage and yielded several striking implications. First, as compared with the SC group, WML was significantly increased in the CHP group and yet was notably reduced in CHP animals after receiving ECSW treatment. Second, compared with SC animals, the integrity of neuN cells was disrupted in CHP animals and yet preserved in CHP-ECSW animals. Third, angiogenesis capacity was attenuated in the CHP group compare to the SC group, but this was significantly reversed in the CHP-ECSW group. The intensities of inflammation and oxidative stress were markedly enhanced in CHP animals than in SC animals, but this was markedly ameliorated in CHP-ECSW animals.
It is well known that WML plays an important role in the initiation and propagation of dementia. Additionally, copious studies have established that CVD is a major contributor for ischemia-related WML and dementia [6,8]. The most important finding in the present study was that WML was notably higher in chronic CHP animals compared to SC animals. Our experimental study, therefore, supports clinical observational studies [6,8], highlighting that bilateral carotid artery stenosis (a scenario of CHP/ischemia) commonly causes WML. Importantly, ECSW therapy protected WM against chronic CHP damage, suggesting that ECSW therapy may have great potential for dementia patients who are refractory or failing to respond to conventional therapies.
An essential finding in the present study was that the number of neuN cells (neurons) was significantly reduced in CHP animals than SC animals. This demonstrates that CHP damaged the myelin sheath as well as the neuron in brain. Our previous study also showed that the number of neuN cells was markedly reduced in rats after acute ischemic stroke, and this was ameliorated with ECSW therapy . In the present study, we identified that ECSW therapy significantly protected the number and integrity of neuN cells. The present study therefore corroborates our previous findings .
Abundant data have previously shown that ischemia/infarction promptly elicits a rigorous inflammatory reaction and oxidative stress that, in turn, cause cellular apoptosis, cellular death and ultimate organ damage and dysfunction [4,17-20,22-24]. Another important finding in the present study was that the inflammatory reaction, oxidative stress and mitochondrial damage were substantially higher in CHP animals than SC animals. Our findings, in addition to strengthening the findings of previous studies [4,17-20,22-24], may also explain why fibrotic, apoptotic, DNA-damage and brain edema (i.e., AQP4) biomarkers within brain tissues were increased in the CHP group than in the SC group. These molecular-cellular perturbations were markedly suppressed in CHP animals after ECSW therapy. Our previous study showed that ECSW therapy significantly attenuated inflammation, the generation of oxidative stress, and cellular apoptosis/death in rats after acute ischaemic stroke, leading to improved neurological function and reduced brain infarct area . In this way, our current findings complement those of our previous study , and provide an important clinically relevant indication that ECSW may be a viable alternative therapeutic option for protecting neurons from CHP-induced apoptosis/death.
Links between ischemia and organ dysfunction have been keenly investigated [4,17-22,24,25]. A principal finding in the present study was that the cellular and molecular expressions of angiogenesis biomarkers, as well as small vessel density, were markedly attenuated in CHP animals compared to SC animals. Given these previous results [4,17-22,24,25], our findings, at least in part, may explain why neurons, myelin sheath and mitochondria were markedly damaged in CHP animals. Of particular importance was that angiogenesis biomarkers and number of small vessels were significantly enhanced in CHP animals after ECSW therapy. Studies have previously established that the fundamental mechanisms underlying improvement of ischemia-related organ dysfunction by ECSW involve inhibition of inflammation and enhancement of angiogenesis [17-21]. Additionally, other studies have identified that restoration of blood flow by means of stem cell or ECSW therapy can protect organs from ischemia-related dysfunction [4,17-22,24]. In this way, our current findings support and reinforce the conclusions of previous reports [4,17-22,24].
This study has limitations. First, any behavioral changes in the rats were not assessed for. Second, WML was measured only with IHC staining but without confirmatory support by brain magnetic resonance imaging findings.
In conclusion, the results of the present study support that ECSW therapy may be an alternative option for preventing CHP-induced brain damage, which is a strong risk factor for development of ischemia-induced dementia.
This study was supported by a program grant from Chang Gung Memorial Hospital, Chang Gung University (Grant number: CMRPG8E0681).