Migration Capacity of Stem Cells from Human Exfoliated Deciduous Teeth Towards Glioma.

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) are a mesenchymal stem cell type and have recently attracted attention for their high proliferative rate, multipotency, and immunosuppressive properties. However, SHED have not yet been investigated for anticancer properties. We therefore investigated whether SHED can be used as a treatment modality, particularly for anti-glioma therapy. METHODS: In vitro, we examined the mobility of SHED and their ability to migrate towards glioma-conditioned medium and specific growth factors secreted by malignant gliomas. In vivo, we transplanted SHED into the left hemisphere of nude mice that had been previously implanted with human malignant glioma U87 cells into the right hemisphere. We assessed whether SHED had tumorigenic potential. RESULTS: SHED exhibited strong migration ability towards malignant glioma in both in vitro and in vivo assays. In vitro, SHED migrated towards glioma-conditioned medium and specific growth factors such as stem cell factor, platelet-derived growth factor BB, C-X-C motif chemokine ligand 12, and vascular endothelial growth factor. SHED were accumulated around tumor cells in the contralateral hemisphere 1 week after transplantation. Moreover, SHED remained in the brains of nude mice 150 days after transplantation. Finally, we verified that SHED had no malignant transformation or engraftment of SHED in the mouse brain. CONCLUSIONS: Our findings indicate that SHED can potentially be applied to track malignant glioma.

Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MßCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MßCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MßCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism.

Prevention Effect of Antiplatelets on Aneurysm Rupture in a Mouse Intracranial Aneurysm Model.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) from intracranial aneurysm rupture results in significant morbidity and mortality. In the present study, we examined the effect of most widely used antiplatelet drugs, aspirin and cilostazol, on aneurysm rupture prevention using a mouse intracranial aneurysm model. MATERIALS AND METHODS: Intracranial aneurysms were induced by a combination of deoxycorticosterone acetate-salt and a single injection of elastase into the cerebrospinal fluid in mice. Treatment with aspirin or cilostazol was started 1 day after aneurysm induction. Aneurysm rupture was detected by neurological symptoms and the presence of intracranial aneurysm with SAH was confirmed by post-mortem examination. RESULTS: Aspirin (10 mg/kg) significantly reduced aneurysm rupture (control:aspirin = 80%:31%, p < 0.05) without affecting the overall incidence of aneurysm formation (60%:62%). Cilostazol (3 mg/kg, 30 mg/kg) did not reduce both rupture rate (control:3 mg/kg:30 mg/kg = 81%:67%:77%) and the overall incidence of aneurysm formation (control:3 mg/kg:30 mg/kg = 72%:71%:76%). Tail vein bleeding time prolonged significantly in both aspirin and cilostazol groups (p < 0.01). CONCLUSION: Aspirin prevented aneurysm rupture in a mouse intracranial aneurysm model, while cilostazol did not. Aspirin, the most frequently used drug for patients with ischemic myocardial and cerebral diseases, is also effective in preventing cerebral aneurysmal rupture.

Neuroprotective effect of neuroserpin in non-tPA-induced intracerebral hemorrhage mouse models.

BACKGROUND: The neuroprotective effects of neuroserpin (NSP) have been well documented in both patients and animal models with cerebral ischemia; however, have never been investigated in hemorrhagic stroke. The aim of this study is to verify the neuroprotection of NSP in the non-tPA-induced intracerebral hemorrhage (ICH) mouse model. METHODS: C57BL/6J male mice (n = 198) were involved in this study. ICH models were established with infusion of autologous blood into the brain parenchyma. We then detected NSP expression in ICH brains by morphological methods and western blotting analysis. We measured the brain water content and detected blood-brain barrier (BBB) permeability to verify the neuroprotective effects of NSP. RESULTS: We found that NSP protein expression was upregulated in ICH models, with a peak at 48 h after ICH induction. NSP local administration reduced the brain edema and the BBB permeability in ICH models. The neurological deficits were also ameliorated. Thus, the neuroprotection of NSP in ICH state was confirmed. Additionally, we also found that the distribution pattern of occludin-expressing cells was obviously changed by the ICH procedure but partly recovered after NSP administration. This finding indicated that protecting and/or repairing the injured vascular endothelial cells may be a potential mechanism involved in NSP neuroprotection, which needs further verification. CONCLUSIONS: Our results supported the fact that NSP may be considered as a potential therapy for ICH for the neuroprotective effects including amelioration of the edema.

Neuroserpin Protects Rat Neurons and Microglia-Mediated Inflammatory Response Against Oxygen-Glucose Deprivation- and Reoxygenation Treatments in an In Vitro Study.

BACKGROUND/AIMS: Neuroserpin (NSP) is known for its neuroprotective role in cerebral ischemic animal models and patients. Our laboratory conducted a series of investigations on the neuroprotection of NSP in different cells in the brain. In the present study, we further observe the effects of NSP on neurons and microglia-mediated inflammatory response following oxygen-glucose deprivation (OGD), and explore possible mechanisms related to neuroprotection of OGD in the central nervous system (CNS). METHODS: Neurons and microglia from neonatal rats were treated with OGD followed by reoxygenation (OGD/R). To confirm the effects of NSP, the neuronal survival, neuronal apoptosis, and lactate dehydrogenase (LDH) release were measured in cultured neurons. Furthermore, the levels of IL-1ß and nitric oxide (NO) release were also detected in cultured microglia. The possible mechanisms for the neuroprotective effect of NSP were explored using Western blot analysis. RESULTS: NSP administration can reverse abnormal variations in neurons and microglia-mediated inflammatory response induced by OGD/R processes. The neuronal survival rate, neuronal apoptosis rate, and LDH release were significantly improved by NSP administration in neurons. Simultaneously, the release of IL-1ß and NO were significantly reduced by NSP in microglia. Western blot showed that the expression of ERK, P38, and JNK was upregulated in microglia by the OGD/R treatment, and these effects were significantly inhibited by NSP. CONCLUSION: These data verified the neuroprotective effects of NSP on neurons and microglia-mediated inflammatory response. Inhibition of the mitogen-activated protein kinase (MAPK) signaling pathways might play a potential role in NSP neuroprotection on microglia-mediated inflammatory response, which needs further verification.

Pseudo-continuous arterial spin labeling reflects vascular density and differentiates angiomatous meningiomas from non-angiomatous meningiomas.

Pseudo-continuous arterial spin labeling (PCASL) can measure tumor blood flow (TBF) reliably. We investigated meningioma TBF using PCASL and assessed for any correlation with histopathological microvascular density (MVD) and the World Health Organization (WHO) classification. Conventional MRI with contrast T1-weighted images and PCASL were acquired with a 3 T scanner before surgery in 25 consecutive patients with meningiomas. Using the PCASL perfusion map, the mean and maximum TBF were calculated from regions of interest placed in the largest cross-sectional plane of each tumor. Tissue sections from 16 patients were stained with CD31 to evaluate MVD and were assigned a WHO classification. The TBFs were statistically compared with MVD and the histopathological meningioma subtypes. There were 16 meningothelial meningiomas, four angiomatous meningiomas, two fibrous meningiomas, one transitional meningioma, and two atypical meningiomas. We observed significant correlation between MVD and both mean and maximum TBF (p < 0.05). The mean and maximum TBF ((mean)TBF, (max)TBF) in angiomatous meningiomas are significantly higher than that in non-angiomatous meningiomas (p < 0.05). PCASL is useful in assessing meningioma vascularity, and in differentiating angiomatous meningiomas from non-angiomatous meningiomas.

Scalp Nerve Block Alleviates Headaches Associated With Sonication During Transcranial Magnetic Resonance-Guided Focused Ultrasound.

BACKGROUND AND OBJECTIVES: In magnetic resonance-guided focused ultrasound (MRgFUS) procedures, headache is a frequent symptom and cause of treatment discontinuation. Herein, we assessed the efficacy of scalp nerve block (SNB) for alleviating headache during MRgFUS procedures. METHODS: The effect of SNB on intraprocedural headache was examined by retrospectively comparing 2 patient cohorts at a single institution. During the study period from April 2020 to February 2022, an SNB protocol for all patients with a skull density ratio ≤0.55 was instituted on October 6, 2021. The number of patients with a skull density ratio ≤0.55 was 34 before the protocol and 36 afterward. Headache intensity was evaluated using a numerical rating scale (NRS) after each sonication. To evaluate the effect of SNB on headache intensity, multiple regression analysis was performed per patient and per sonication. In the per-patient analysis, the effect of SNB was evaluated using the maximum NRS, mean NRS, and NRS at the first ultrasound exposure that reached 52.5°C. In the per-sonication analysis, the effect of SNB was evaluated not only for the entire sonication but also for sonications classified into ≤9999 J, 10 000 to 29 999 J, and ≥30 000 J energy doses. RESULTS: With SNB, headache alleviation was observed in the NRS after the first sonication that reached 52.5°C in each patient (ß = -2.40, 95% CI -4.05 to -0.758, P = .00499), in the NRS when all sonications were evaluated (ß = -0.647, 95% CI -1.19 to -0.106, P = .0201), and in the NRS when all sonications were classified into 10 000 to 29 999 J (ß = -1.83, 95% CI -3.17 to -0.485, P = .00889). CONCLUSION: SNB significantly reduced headache intensity during MRgFUS, especially that caused by sonication with a moderate-energy dose. These findings suggest that scalp nerves play a role in headache mechanisms during MRgFUS.

Evaluation of tumor blood flow after feeder embolization in meningiomas by arterial spin-labeling perfusion magnetic resonance imaging.

Preoperative embolization changes the amount of blood flow and pattern of flow distribution in meningioma. Tumor blood flow was investigated in eight meningioma patients before and after embolization using arterial spin-labeling (ASL) perfusion imaging. Although blood flow was significantly reduced in the whole tumor after embolization, changes in flow distribution patterns varied from one case to another. The findings suggest that evaluation of post-embolization tumor blood flow by ASL perfusion imaging would be useful in the surgical planning of meningioma.

Potent bystander effect and tumor tropism in suicide gene therapy using stem cells from human exfoliated deciduous teeth.

Herpes simplex virus thymidine kinase (HSVTK)/ganciclovir (GCV) suicide gene therapy has a long history of treating malignant gliomas. Recently, stem cells from human exfoliated deciduous teeth (SHED), which are collected from deciduous teeth and have excellent harvestability, ethical aspects, and self-renewal, have been attracting attention mainly in the field of gene therapy. In the present study, we assessed SHED as a novel cellular vehicle for suicide gene therapy in malignant gliomas, as we have previously demonstrated with various cell types. SHED was transduced with the HSVTK gene (SHEDTK). In vitro experiments showed a significant bystander effect between SHEDTK and glioma cell lines in coculture. Furthermore, apoptotic changes caused by caspase 3/7 activation were simultaneously observed in SHEDTK and glioma cells. Mice implanted with a mixture of U87 and SHEDTK and treated with intraperitoneal GCV survived for longer than 100 days. Additionally, tumors in treatment model mice were significantly reduced in size during the treatment period. SHEDTK implanted at the contralateral hemisphere migrated toward the tumor crossing the corpus callosum. These results suggested that SHEDTK-based suicide gene therapy has potent tumor tropism and a bystander-killing effect, potentially offering a new promising therapeutic modality for malignant gliomas.

Immunohistochemical analysis-based proteomic subclassification of newly diagnosed glioblastomas.

Recent gene expression and copy number profilings of glioblastoma multiforme (GBM) by The Cancer Genome Atlas (TCGA) Research Network suggest the existence of distinct subtypes of this tumor. However, these approaches might not be easily applicable in routine clinical practice. In the current study, we aimed to establish a proteomics-based subclassification of GBM by integrating their genomic and epigenomic profiles. We subclassified 79 newly diagnosed GBM based on expression patterns determined by comprehensive immunohistochemical observation in combination with their DNA copy number and DNA methylation patterns. The clinical relevance of our classification was independently validated in TCGA datasets. Consensus clustering identified the four distinct GBM subtypes: Oligodendrocyte Precursor (OPC) type, Differentiated Oligodendrocyte (DOC) type, Astrocytic Mesenchymal (AsMes) type and Mixed type. The OPC type was characterized by highly positive scores of Olig2, PDGFRA, p16, p53 and synaptophysin. In contrast, the AsMes type was strongly associated with strong expressions of nestin, CD44 and podoplanin, with a high glial fibrillary acidic protein score. The median overall survival of OPC-type patients was significantly longer than that of the AsMes-type patients (19.9 vs 12.8 months). This finding was in agreement with the Oncomine analysis of TCGA datasets, which revealed that PDGFRA and Olig2 were favorable prognostic factors and podoplanin and CD44 were associated with a poor clinical outcome. This is the first study to establish a subclassification of GBM on the basis of immunohistochemical analysis. Our study will shed light on personalized therapies that might be feasible in daily neuropathological practice.

Effects of ghrelin on the intracellular calcium concentration in rat aorta vascular smooth muscle cells.

BACKGROUND/AIMS: Ghrelin has been regarded as a cardioprotective factor with complicated mechanisms. Whether ghrelin is vasodilative or vasoconstrictive in nature is controversial, and the effects of ghrelin on intracellular calcium concentration are still unclear. To explore the mechanisms involved in the vasoactive regulation of ghrelin at the cellular level, we investigated the effects of ghrelin on calcium concentrations in rat aorta vascular smooth muscle cells (VSMCs). METHODS: We obtained VSMCs via cell culture and stained the cells with Furo-2 AM. Western blotting was used to verify growth hormone secretagogue receptor (GHS-R1a) expression in VSMCs. The intracellular calcium variations affected by ghrelin and the interactions of ghrelin with angiotensin II (AngII), Sq22536, and potassium chloride (KCl) were observed using a calcium imaging and analysis system. RESULTS: Western blotting revealed good GHS-R1a expression in VSMCs. The most prominent finding in the present study was that ghrelin inhibited the AngII-induced increase in the calcium concentration. This inhibition was reversed by the adenylate cyclase inhibitor Sq22536 and the GHS-R1a antagonist (D-Lys(3))- GHRP-6. This finding revealed the potential vasodilative effects of ghrelin at the cellular level. We did not observe any effects of ghrelin on intracellular calcium concentrations in resting VSMCs or the increase of calcium concentration induced by KCl. CONCLUSION: Ghrelin inhibited the increase in the intracellular calcium concentration of rat aorta VSMCs induced by AngII, which may depend on the activation of the cAMP/PKA pathway.

Efficacy of HSV-TK/GCV system suicide gene therapy using SHED expressing modified HSV-TK against lung cancer brain metastases.

Lung cancer is one of the most common cancers, and the number of patients with intracranial metastases is increasing. Previously, we developed an enzyme prodrug suicide gene therapy based on the herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system using various mesenchymal stem cells to induce apoptosis in malignant gliomas through bystander killing effects. Here, we describe stem cells from human exfoliated deciduous teeth (SHED) as gene vehicles of the TK/GCV system against a brain metastasis model of non-small cell lung cancer (NSCLC). We introduced the A168H mutant TK (TKA168H) into SHED to establish the therapeutic cells because of the latent toxicity of wild type. SHED expressing TKA168H (SHED-TK) exhibited chemotaxis to the conditioned medium of NSCLC and migrated toward implanted NSCLC in vivo. SHED-TK demonstrated a strong bystander effect in vitro and in vivo and completely eradicated H1299 NSCLC in the brain. SHED-TK cells implanted intratumorally followed by GCV administration significantly suppressed the growth of H1299 and improved survival time. These results indicate that the TKA168H variant is suitable for establishing therapeutic cells and that intratumoral injection of SHED-TK followed by GCV administration may be a useful strategy for therapeutic approaches.

Transient Global Amnesia Following Coil Embolization for a Basilar-tip Aneurysm: Case Reports of Two Patients.

Transient global amnesia (TGA) can be caused by medications, ischemia, metabolic abnormalities, and seizures. We describe two cases of TGA following coil embolization for a basilar-tip aneurysm. A 73-year-old woman developed transient acute anterograde amnesia after coil embolization for a basilar-tip aneurysm. Diffusion-weighted imaging (DWI) revealed an ischemic lesion in the anterior nucleus of the thalamus. A 67-year-old woman developed transient acute amnesia after a stent-assisted coil embolization of a basilar-tip aneurysm. A DWI showed ischemic lesions in the anterior nucleus of the thalamus. Any ischemic changes to areas of the anterior nucleus that are fed by the thalamoperforating and premammillary arteries should be considered in a differential diagnosis for TGA in patients who have undergone coil embolization for a posterior circulation cerebral aneurysm.

Finite element method for nerve root decompression in minimally invasive endoscopic spinal surgery.

INTRODUCTION: Diagnosis is the key to improving spinal surgery outcomes. Improvements in the diagnosis of radiculopathy have created new indications for full-endoscopic spine surgery. We assessed the finite element method (FEM) to visualize and digitize lesions not detected by conventional diagnostic imaging. METHODS: We used FEM in two patients: a lumbar patient and a cervical patient. The lumbar patient was a 67-year-old woman with a history of rheumatoid arthritis; she also had osteoporosis and pulmonary fibrosis. She had left L3 radiculopathy due to an L3 vertebral fracture. The cervical patient was a 61-year-old woman with left C6 radiculopathy due to C5-C6 disc herniation. We performed full endoscopic foraminotomy per the patients's request. Based on preoperative and postoperative CT Digital Imaging and Communications in Medicine data of 0.5-mm slices, 3-D imaging data were reproduced, and kinetic simulation of FEM was performed. RESULTS: Postoperatively, both patients' radiculopathy disappeared, improving their activities of daily living and enabling them to walk and work. Also, the total contact area and maximum contact pressure of the nerve tissue decreased from 30% to 80% and from 33% to 67%, respectively. CONCLUSIONS: A new method for perioperative evaluation and simulation, FEM can be to visualize and digitize the conditions of the lesion causing radiculopathy. FEM that can overcome both time and economic constraints in routine clinical practice is needed.

Increased anteroventral striatal dopamine transporter and motor recovery after subthalamic deep brain stimulation in Parkinson's disease.

OBJECTIVE: Subthalamic nucleus deep brain stimulation (STN-DBS) in Parkinson's disease is effective; however, its mechanism is unclear. To investigate the degree of neuronal terminal survival after STN-DBS, the authors examined the striatal dopamine transporter levels before and after treatment in association with clinical improvement using PET with [11C]2ß-carbomethoxy-3ß-(4-fluorophenyl)tropane ([11C]CFT). METHODS: Ten patients with Parkinson's disease who had undergone bilateral STN-DBS were scanned twice with [11C]CFT PET just before and 1 year after surgery. Correlation analysis was conducted between [11C]CFT binding and off-period Unified Parkinson's Disease Rating Scale (UPDRS) scores assessed preoperatively and postoperatively. RESULTS: [11C]CFT uptake reduced significantly in the posterodorsal putamen contralateral to the parkinsonism-dominant side after 1 year; however, an increase was noted in the contralateral anteroventral putamen and ipsilateral ventral caudate postoperatively (p < 0.05). The percentage increase in [11C]CFT binding was inversely correlated with the preoperative binding level in the bilateral anteroventral putamen, ipsilateral ventral caudate, contralateral anterodorsal putamen, contralateral posteroventral putamen, and contralateral nucleus accumbens. The percentage reduction in UPDRS-II score was significantly correlated with the percentage increase in [11C]CFT binding in the ipsilateral anteroventral putamen (p < 0.05). The percentage reduction in UPDRS-III score was significantly correlated with the percentage increase in [11C]CFT binding in the ipsilateral anteroventral putamen, ventral caudate, and nucleus accumbens (p < 0.05). CONCLUSIONS: STN-DBS increases dopamine transporter levels in the anteroventral striatum, which is correlated with the motor recovery and possibly suggests the neuromodulatory effect of STN-DBS on dopaminergic terminals in Parkinson's disease patients. A preoperative level of anterior striatal dopamine transporter may predict reserve capacity of STN-DBS on motor recovery.

Feasibility of Mechanical Thrombectomy for Acute Ischemic Stroke Patients Aged 90 Years or Older Compared to Younger Patients.

Mechanical thrombectomy (MT) is a proven treatment for acute ischemic stroke (AIS). However, the efficacy of this treatment is uncertain for very elderly patients. This study aimed to investigate the safety and effectiveness of MT in 90 years or older patients compared with younger patients. We retrospectively reviewed AIS patients treated with MT between October 2018 and June 2020 in our institution. Patients were divided into two groups: aged ≥90 and <90 years. We compared the following factors: functional outcome at discharge, in-hospital death, successful recanalization, and complications. Multivariate logistic regression analysis for the good functional outcome was performed. In consideration of pre-stroke basic activities of very elderly patients, we defined the good functional outcome as modified Rankin Scale (mRS) 0-3. In all, 66 patients were included, and 19 patients (28%) were ≥90 years old. Pre-stoke mRS was higher in ≥90-year-old patients (p = 0.01). In ≥90-year-old patients, we achieved successful recanalization in 17 patients (90%), and only one patient experienced hemorrhagic complication related with the procedure. The good functional outcome (mRS: 0-3) at discharge were six patients (32%) in ≥90 years old versus 19 patients (40%) in <90 years old (p = 0.6). Three patients died in hospital in each group (16% versus 6%) (p = 0.3). Only the stroke severity was negatively related with the good functional outcome in a multivariate analysis. In conclusion, for ≥90-year-old patients compared with younger patients, MT is an equally feasible therapy. Patients should not be excluded from MT based on age alone.

Comparing impacts of metal contamination on macroinvertebrate and fish assemblages in a northern Japanese river.

Researchers have long assessed the ecological impacts of metals in running waters, but few such studies investigated multiple biological groups. Our goals in this study were to assess the ecological impacts of metal contamination on macroinvertebrates and fishes in a northern Japanese river receiving treated mine discharge and to evaluate whether there was any difference between the metrics based on macroinvertebrates and those based on fishes in assessing these impacts. Macroinvertebrate communities and fish populations were little affected at the downstream contaminated sites where concentrations of Cu, Zn, Pb, and Cd were 0.1-1.5 times higher than water-quality criteria established by the U.S. Environmental Protection Agency. We detected a significant reduction in a few macroinvertebrate metrics such as mayfly abundance and the abundance of heptageniid mayflies at the two most upstream contaminated sites with metal concentrations 0.8-3.7 times higher than the water-quality criteria. There were, however, no remarkable effects on the abundance or condition factor of the four dominant fishes, including masu salmon (Oncorhynchus masou). These results suggest that the richness and abundance of macroinvertebrates are more sensitive to metal contamination than abundance and condition factor of fishes in the studied river. Because the sensitivity to metal contamination can depend on the biological metrics used, and fish-based metrics in this study were limited, it would be valuable to accumulate empirical evidence for ecological indicators sensitive to metal contamination within and among biological groups to help in choosing which groups to survey for general environmental impact assessments in metal-contaminated rivers.