Gadolinium Retention and Nephrotoxicity in a Mouse Model of Acute Ischemic Stroke: Linear Versus Macrocyclic Agents.

BACKGROUND: Gadolinium (Gd)-based contrast agents (GBCAs) have been widely used for acute ischemic stroke (AIS) patients. GBCAs or AIS alone may cause the adverse effects on kidney tissue, respectively. However, whether GBCAs and AIS would generate a synergistic negative effect remains undefined. PURPOSE: To evaluate synergistic negative effects of AIS and GBCAs on renal tissues in a mouse model of AIS, and to compare the differences of these negative effects between linear and macrocyclic GBCAs. STUDY TYPE: Animal study. ANIMAL MODEL: Seventy-two healthy mice underwent transient middle cerebral artery occlusion (tMCAO) and sham operation to establish AIS and sham model (N = 36/model). 5.0 mmol/kg GBCAs (gadopentetate or gadobutrol) or 250 µL saline were performed at 4.5 hours and 1 day after model establishing (N = 12/group). ASSESSMENT: Inductively coupled plasma mass spectrometry (ICP-MS) was performed to detect Gd concentrations. Serum biochemical analyzer was performed to measure the serum creatinine (Scr), uric acid (UA), and blood urea nitrogen (BUN). Pathological staining was performed to observe tubular injury, cell apoptosis, mesangial hyperplasia, and interstitial fibrosis. STATISTICAL TESTS: Two-way analysis of variances with post hoc Sidak's tests and independent-samples t-tests were performed. A P-value <0.05 was considered statistically significant. RESULTS: AIS groups showed higher Gd concentration than sham group on day 1 p.i. regardless of gadopentetate or gadobutrol used. Increased total Gd concentration was also found in AIS + gadopentetate group compared with the sham group on day 28 p.i. Significantly higher rates for renal dysfunction, higher tubular injury scores, and higher numbers of apoptotic cells on days 1 or 28 p.i. were found for AIS mice injected with GBCA. AIS + gadopentetate group displayed more severe renal damage than the AIS + gadobutrol group. DATA CONCLUSION: AIS and GBCAs may cause increased total Gd accumulation and nephrotoxicity in a mouse, especially linear GBCAs were used. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 4.

Lipidomic analysis identifies long-chain acylcarnitine as a target for ischemic stroke.

INTRODUCTION: Lipid metabolism dysfunction is widely involved in the pathological process of acute ischemic stroke (AIS). The coordination of lipid metabolism between neurons and astrocytes is of great significance. However, the full scope of lipid dynamic changes and the function of key lipids during AIS remain unknown. Hence, identifying lipid alterations and characterizing their key roles in AIS is of great importance. METHODS: Untargeted and targeted lipidomic analyses were applied to profile lipid changes in the ischemic penumbra and peripheral blood of transient middle cerebral artery occlusion (tMCAO) mice as well as the peripheral blood of AIS patients. Infarct volume and neurological deficits were assessed after tMCAO. The cell viability and dendritic complexity of primary neurons were evaluated by CCK8 assay and Sholl analysis. Seahorse, MitoTracker Green, tetramethyl rhodamine methyl ester (TMRM), 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSOX were used as markers of mitochondrial health. Fluorescent and isotopic free fatty acid (FFA) pulse-chase assays were used to track FFA flux in astrocytes. RESULTS: Long-chain acylcarnitines (LCACs) were the lipids with the most dramatic changes in the ischemic penumbra and peripheral blood of tMCAO mice. LCACs were significantly elevated on admission in AIS patients and associated with poor outcomes in AIS patients. Increasing LCACs through a bolus administration of palmitoylcarnitine amplified stroke injury, while decreasing LCACs by overexpressing carnitine palmitoyltransferase 2 (CPT2) ameliorated stroke injury. Palmitoylcarnitine aggravated astrocytic mitochondrial damage after OGD/R, while CPT2 overexpression in astrocytes ameliorated cocultured neuron viability. Further study revealed that astrocytes stimulated by OGD/R liberated FFAs from lipid droplets into mitochondria to form LCACs, resulting in mitochondrial damage and lowered astrocytic metabolic support and thereby aggravated neuronal damage. CONCLUSION: LCACs could accumulate and damage neurons by inducing astrocytic mitochondrial dysfunction in AIS. LCACs play a crucial role in the pathology of AIS and are novel promising diagnostic and prognostic biomarkers for AIS.

Gadolinium retention in the ischemic cerebrum: Implications for pain, neuron loss, and neurological deficits.

PURPOSE: To investigate the effects of gadolinium (Gd) retention of macrocyclic (gadobutrol) or linear (gadopentetate) Gd-based contrast agents (GBCAs) on neuron loss, neurological deficits, and sensory behavior in mice with or without stroke. METHODS: Ninety C57BL/6 mice underwent sham (n = 36) or transient middle cerebral artery occlusion (tMCAO) (n = 54) surgery and then received intraperitoneal injections of 5.0 mmol/kg gadobutrol, 5.0 mmol/kg gadopentetate or saline (10 ml/kg/administration) per day for 3 consecutive days. The Gd concentration in the ischemic cerebrum was quantified by inductively coupled plasma mass spectrometry on Day 1 and Day 28 after the last injection (post-injection, p. i.). Neuron loss, glia activation and neurological deficits were assessed on Day 1 and 28 p. i. Sensory behavior was also assessed on Day 28 p. i. RESULTS: Gd concentrations were higher in the brains of tMCAO mice than in those of sham mice on Days 1 p. i. of both GBCAs (gadobutrol, p &lt; 0.05; gadopentetate, p &lt; 0.001) and 28 p. i of gadopentetate. (p &lt; 0.001). Sham or tMCAO mice injected with GBCAs showed no significant difference in neuron loss, glia activation, neurological deficits, brain atrophy, or hippocampus-dependent memory (all p &gt; 0.05). Both gadobutrol and gadopentetate induced mechanical and heat hyperalgesia in sham mice (all p &lt; 0.05). However, mechanical hyperalgesia but rather heat hyperalgesia was found in tMCAO mice with the highest force tested (1.0 g) and statistically significant in both paws (right and left) with gadopentetate only (p &lt; 0.05). CONCLUSIONS: Neither gadobutrol nor gadopentetate worsened neuron loss, glia activation, brain atrophy, neurological deficits, or hippocampus-dependent memory after tMCAO. However, GBCA administration induced mechanical hyperalgesia in sham and tMCAO mice although in the same level, which may be an important consideration for patients with central post-stroke pain and those who are sensitive to pain and about to receive multiple GBCA administrations.

Longitudinal Multiparametric MRI Assessment of Irradiated Salivary Gland in a Rat Model: Correlated With Histological Findings.

BACKGROUND: Several magnetic resonance imaging (MRI) sequences have been applied to assess injured glands but without histological validation. PURPOSE: To evaluate longitudinal changes in multiparametric MRI (mp-MRI) of irradiated salivary glands in a rat model and investigate correlations between mp-MRI and histological findings. STUDY TYPE: Prospective. ANIMAL MODEL: Submandibular glands of 36 rats were radiated using a single dose of 15 Gy X-ray (irradiation [IR] group), and 6 other rats were enrolled into sham-IR group. mp-MRI were scanned 1 day after sham-IR (n = 6), or 1, 2, 4, 8, 12, 24 weeks after IR (n = 36, 6 per subgroup). FIELD STRENGTH/SEQUENCE: A 3.0-T/Diffusion-weighted imaging (DWI), readout-segmented echo-planar imaging (EPI) sequence; intravoxel incoherent motion DWI, single-shot EPI sequence; T1 mapping, dual-flip-angle gradient-echo sequence with volumetric interpolated breath-hold examination; T2 mapping, turbo spin-echo sequence. ASSESSMENT: Parameters including apparent diffusion coefficient (ADC), pure diffusion coefficient (D), pseudo-diffusion coefficient (D* ), perfusion fraction (f), T1 and T2 value were obtained. Histological examinations, including hematoxylin and eosin staining (for acinar cell fraction [AC%] detection), Masson's trichrome staining (for degree of fibrosis [F%] determination) and CD34-immunohistochemical staining (for microvessel density [MVD] calculation), were performed at corresponding time points. STATISTICAL TESTS: One-way analysis of variance was used to compare the mp-MRI and histological parameters among different groups. Spearman correlation analysis was applied to determine the correlation between mp-MRI and histological parameters. Two-sided P ≤ 0.05 was considered statistically significant. RESULTS: Changes of mp-MRI parameters (ADC, D, D* , f, T1, T2) and histological results (AC%, F%, MVD) among the seven groups were all significant. ADC, D, and T2 values negatively correlated with AC% (ADC, r = -0.728; D, r = -0.773; T2, r = -0.600), f positively correlated with MVD (r = 0.496), and T1 values positively correlated with F% (r = 0.714). DATA CONCLUSION: mp-MRI might be able to noninvasively and quantitatively evaluate the dynamic pathological changes within the irradiated salivary glands. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 2.

Ischemic Stroke Increased Gadolinium Deposition in the Brain and Aggravated Astrocyte Injury After Gadolinium-Based Contrast Agent Administration: Linear Versus Macrocyclic Agents.

BACKGROUND: Gadolinium (Gd)-based contrast agents (GBCAs) have been widely used in MRI. However, several studies have reported Gd deposition in the brain, which has raised concerns about safety. PURPOSE: To investigate the effects of ischemic stroke on Gd deposition in the brain after repeated administration of linear or macrocyclic GBCAs and to determine whether GBCAs aggravate astrocyte injury after stroke. STUDY TYPE: Animal study. ANIMAL MODEL: Twenty-seven male Sprague-Dawley rats were randomized to an exposure group (n = 24) and a healthy control group (n = 3). Half of the exposure group (n = 12) underwent transient middle cerebral artery occlusion (tMCAO) and half (n = 12) had a sham procedure. In each subgroup (tMCAO or sham), the rats had repeated gadopentetate (n = 6) or gadobutrol (n = 6) injections. Oxygen-glucose deprivation and reoxygenation (OGD/R) was used as an in vitro model of stroke. ASSESSMENT: On day 3 and day 28 after the last injection (p.i.), the Gd concentration in the cerebrum was quantified by inductively coupled plasma mass spectrometry. Cell viability, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were analyzed in vitro. STATISTICAL TESTS: One-way analysis of variance and two-sample t-tests were performed. RESULTS: The Gd concentration in the ipsilateral hemisphere homogenates of tMCAO group was significantly higher than that in the brain homogenates of the sham group on day 3 p.i. of either gadobutrol (0.065 ± 0.006 vs. 0.042 ± 0.007 µg/g, P < 0.05) or gadopentetate (0.093 ± 0.010 vs. 0.069 ± 0.008 µg/g, P < 0.05). Increased Gd deposition was also found in the ipsilateral hemisphere homogenates of the tMCAO group compared with the brain homogenates of the sham group on day 28 p.i. of gadopentetate (0.075 ± 0.012 vs. 0.044 ± 0.003 µg/g, P < 0.05), but not gadobutrol (0.012 ± 0.007 vs. 0.010 ± 0.001 µg/g, P = 0.80). The Gd concentration in the ipsilateral hemisphere in the tMCAO group was significantly higher for gadopentetate than gadobutrol on both day 3 p.i. (0.085 ± 0.006 vs. 0.049 ± 0.005 µg/g, P < 0.05) and day 28 p.i (0.075 ± 0.012 vs. 0.012 ± 0.007 µg/g, P < 0.05). Additionally, compared with gadobutrol, gadopentetate decreased viability, increased ROS accumulation, and decreased MMP in OGD/R-induced astrocytes (all P < 0.05). DATA CONCLUSION: Administration of GBCAs after an animal model of ischemic stroke increased Gd deposition in the brain and aggravated astrocyte injury. The effect of gadopentetate appeared to be more pronounced than that of gadobutrol.

A Canine Model of Hemorrhagic Transformation Using Recombinant Tissue Plasminogen Activator Administration After Acute Ischemic Stroke.

Early reperfusion of occluded arteries via recombinant tissue plasminogen activator (rtPA) administration is considered to be an effective strategy for the treatment of acute ischemic stroke. However, delayed administration of rtPA may cause severe hemorrhagic transformation (HT) and undesirable neurological outcomes. The current study aims to establish a canine HT model using rtPA administration and to investigate the potential mechanisms underlying HT. Following anesthesia, two autologous clots were injected into the middle cerebral artery (MCA) to induce ischemic stroke. To induce reperfusion, rtPA (2 mg/kg) was administrated intravenously 4.5 h after the establishment of stroke. The occurrence of HT was determined by computed tomography (CT) and by pathological assessment. Transmission electron microscopy was utilized to assess blood-brain barrier (BBB) damage. The expression of matrix metalloprotein 9 (MMP-9) was analyzed by enzyme linked immunosorbent assay (ELISA), immunofluorescence (IF), and western blot. Administration of rtPA 4.5 h after stroke induced reperfusion in 73.9% of the canines, caused evident HT, and did not improve neurological outcomes compared to canines that did not receive rtPA. There was a significant increase in expression of MMP-9 after rtPA administration, accompanied by BBB disruption. We have established a canine HT model that closely mimics human HT by using rtPA administration after the induction of middle cerebral artery occlusion (MCAO) with autologous clots. Our data suggest that a potential mechanism underlying rtPA-caused HT may be related to BBB dysfunction induced by an increase in MMP-9 expression.

Hypoxic conditioned medium derived from bone marrow mesenchymal stromal cells protects against ischemic stroke in rats.

In recent years, studies have shown that the secretome of bone marrow mesenchymal stromal cells (BMSCs) contains many growth factors, cytokines, and antioxidants, which may provide novel approaches to treat ischemic diseases. Furthermore, the secretome may be modulated by hypoxic preconditioning. We hypothesized that conditioned medium (CM) derived from BMSCs plays a crucial role in reducing tissue damage and improving neurological recovery after ischemic stroke and that hypoxic preconditioning of BMSCs robustly improves these activities. Rats were subjected to ischemic stroke by middle cerebral artery occlusion and then intravenously administered hypoxic CM, normoxic CM, or Dulbecco modified Eagle medium (DMEM, control). Cytokine antibody arrays and label-free quantitative proteomics analysis were used to compare the differences between hypoxic CM and normoxic CM. Injection of normoxic CM significantly reduced the infarct area and improved neurological recovery after stroke compared with administering DMEM. These outcomes may be associated with the attenuation of apoptosis and promotion of angiogenesis. Hypoxic preconditioning significantly enhanced these therapeutic effects. Fourteen proteins were significantly increased in hypoxic CM compared with normoxic CM as measured by cytokine arrays. The label-free quantitative proteomics analysis revealed 163 proteins that were differentially expressed between the two groups, including 107 upregulated proteins and 56 downregulated proteins. Collectively, our results demonstrate that hypoxic CM protected brain tissue from ischemic injury and promoted functional recovery after stroke in rats and that hypoxic CM may be the basis of a potential therapy for stroke patients.

The CD40/CD40L system regulates rat cerebral microvasculature after focal ischemia/reperfusion via the mTOR/S6K signaling pathway.

OBJECTIVE: The role of CD40/CD40 ligand (CD40L) in microvascular thrombosis is now widely accepted. However, the exact mechanisms linking the CD40/CD40L system and the soluble form of CD40L (sCD40L) with microvascular thrombosis are currently a topic of intensive research. The objective of this study was to assess the potential mechanisms in CD40/CD40L system-regulated microvascular thrombosis after focal ischemia/reperfusion (I/R). METHODS: Rats were subjected to 60-min transient middle cerebral artery occlusion (MCAO). The experiments were divided into three groups: sham operation, MCAO, and MCAO + CD40 antagonist. Dynamic changes of serum-free sCD40L levels for 0, 1, 3, 5, 6, and 12 h by ELISA detecting kit after focal I/R were observed, and the CD40 expression levels in both platelet surface and vascular endothelial cell surface were measured by flow cytometry and immunofluorescence, respectively. Cerebral infarct volume was analyzed 12 h after reperfusion. mTOR/S6K signaling was determined by Western blot. RESULTS: A comparison of thrombus formation between MCAO and CD40 antagonist treatment rats revealed a role for CD40 and/or CD40L in the inflammation-enhanced thrombosis responses in both of the platelet and vascular endothelial cell. MCAO rats yielded an acceleration of thrombus formation that was accompanied by increased CD40 levels in serum. The brain infarction was significantly decreased in CD40 antagonist treatment group compared to MCAO model group. The mTOR/S6K signaling was activated in MACO model than that of CD40 antagonist treatment group. CONCLUSIONS: Our findings indicate that CD40/CD40L system contributes to microvascular thrombosis and brain infarction induced by MCAO and reperfusion. The mTOR/S6K signaling pathway is involved in the regulation of cerebral microvasculature after focal I/R by CD40/CD40L. ABBREVIATIONS: AKT: protein kinase B; CD40L: CD40 ligand; CSF: cerebrospinal fluid; FITC: fluorescein isothiocyanate; I/R: ischemia/reperfusion; MCAO: middle cerebral artery occlusion; mTOR: mechanistic target of rapamycin; PE: P-phycoerythrin; sCD40L: soluble form of CD40L; TNF-a: tumor necrosis factor-alpha; WT: wild type.