Spatially selective stimulation of the pig vagus nerve to modulate target effect versus side effect.

Electrical stimulation of the cervical vagus nerve using implanted electrodes (VNS) is FDA-approved for the treatment of drug-resistant epilepsy, treatment-resistant depression, and most recently, chronic ischemic stroke rehabilitation. However, VNS is critically limited by the unwanted stimulation of nearby neck muscles-a result of non-specific stimulation activating motor nerve fibers within the vagus. Prior studies suggested that precise placement of small epineural electrodes can modify VNS therapeutic effects, such as cardiac responses. However, it remains unclear if placement can alter the balance between intended effect and limiting side effect. We used an FDA investigational device exemption approved six-contact epineural cuff to deliver VNS in pigs and quantified how epineural electrode location impacts on- and off-target VNS activation. Detailed post-mortem histology was conducted to understand how the underlying neuroanatomy impacts observed functional responses. Here we report the discovery and characterization of clear neuroanatomy-dependent differences in threshold and saturation for responses related to both effect (change in heart rate) and side effect (neck muscle contractions). The histological and electrophysiological data were used to develop and validate subject-specific computation models of VNS, creating a well-grounded quantitative framework to optimize electrode location-specific activation of nerve fibers governing intended effect versus unwanted side effect.

In vivo Visualization of Pig Vagus Nerve "Vagotopy" Using Ultrasound.

Background: Placement of the clinical vagus nerve stimulating cuff is a standard surgical procedure based on anatomical landmarks, with limited patient specificity in terms of fascicular organization or vagal anatomy. As such, the therapeutic effects are generally limited by unwanted side effects of neck muscle contractions, demonstrated by previous studies to result from stimulation of (1) motor fibers near the cuff in the superior laryngeal and (2) motor fibers within the cuff projecting to the recurrent laryngeal. Objective: Conventional non-invasive ultrasound, where the transducer is placed on the surface of the skin, has been previously used to visualize the vagus with respect to other landmarks such as the carotid and internal jugular vein. However, it lacks sufficient resolution to provide details about the vagus fascicular organization, or detail about smaller neural structures such as the recurrent and superior laryngeal branch responsible for therapy limiting side effects. Here, we characterize the use of ultrasound with the transducer placed in the surgical pocket to improve resolution without adding significant additional risk to the surgical procedure in the pig model. Methods: Ultrasound images were obtained from a point of known functional organization at the nodose ganglia to the point of placement of stimulating electrodes within the surgical window. Naïve volunteers with minimal training were then asked to use these ultrasound videos to trace afferent groupings of fascicles from the nodose to their location within the surgical window where a stimulating cuff would normally be placed. Volunteers were asked to select a location for epineural electrode placement away from the fascicles containing efferent motor nerves responsible for therapy limiting side effects. 2-D and 3-D reconstructions of the ultrasound were directly compared to post-mortem histology in the same animals. Results: High-resolution ultrasound from the surgical pocket enabled 2-D and 3-D reconstruction of the cervical vagus and surrounding structures that accurately depicted the functional vagotopy of the pig vagus nerve as confirmed via histology. Although resolution was not sufficient to match specific fascicles between ultrasound and histology 1 to 1, it was sufficient to trace fascicle groupings from a point of known functional organization at the nodose ganglia to their locations within the surgical window at stimulating electrode placement. Naïve volunteers were able place an electrode proximal to the sensory afferent grouping of fascicles and away from the motor nerve efferent grouping of fascicles in each subject (n = 3). Conclusion: The surgical pocket itself provides a unique opportunity to obtain higher resolution ultrasound images of neural targets responsible for intended therapeutic effect and limiting off-target effects. We demonstrate the increase in resolution is sufficient to aid patient-specific electrode placement to optimize outcomes. This simple technique could be easily adopted for multiple neuromodulation targets to better understand how patient specific anatomy impacts functional outcomes.

Changes in spinal cord hemodynamics reflect modulation of spinal network with different parameters of epidural stimulation.

In this study functional ultrasound (fUS) imaging has been implemented to explore the local hemodynamics response induced by electrical epidural stimulation and to study real-time in vivo functional changes of the spinal cord, taking advantage of the superior spatiotemporal resolution provided by fUS. By quantifying the hemodynamics and electromyographic response features, we tested the hypothesis that the temporal hemodynamics response of the spinal cord to electrical epidural stimulation could reflect modulation of the spinal circuitry and accordingly respond to the changes in parameters of electrical stimulation. The results of this study for the first time demonstrate that the hemodynamics response to electrical stimulation could reflect a neural-vascular coupling of the spinal cord. Response in the dorsal areas to epidural stimulation was significantly higher and faster compared to the response in ventral spinal cord. Positive relation between the hemodynamics and the EMG responses was observed at the lower frequencies of epidural stimulation (20 and 40 Hz), which according to our previous findings can facilitate spinal circuitry after spinal cord injury, compared to higher frequencies (200 and 500 Hz). These findings suggest that different mechanisms could be involved in spinal cord hemodynamics changes during different parameters of electrical stimulation and for the first time provide the evidence that neural-vascular coupling of the spinal cord circuitry could be related to specific organization of spinal cord vasculature and hemodynamics.

Sources of off-target effects of vagus nerve stimulation using the helical clinical lead in domestic pigs.

Objective: Clinical data suggest that efficacious vagus nerve stimulation (VNS) is limited by side effects such as cough and dyspnea that have stimulation thresholds lower than those for therapeutic outcomes. VNS side effects are putatively caused by activation of nearby muscles within the neck, via direct muscle activation or activation of nerve fibers innervating those muscles. Our goal was to determine the thresholds at which various VNS-evoked effects occur in the domestic pig­an animal model with vagus anatomy similar to human­using the bipolar helical lead deployed clinically. Approach: Intrafascicular electrodes were placed within the vagus nerve to record electroneurographic (ENG) responses, and needle electrodes were placed in the vagal-innervated neck muscles to record electromyographic (EMG) responses. Main results: Contraction of the cricoarytenoid muscle occurred at low amplitudes (~0.3 mA) and resulted from activation of motor nerve fibers in the cervical vagus trunk within the electrode cuff which bifurcate into the recurrent laryngeal branch of the vagus. At higher amplitudes (~1.4 mA), contraction of the cricoarytenoid and cricothyroid muscles was generated by current leakage outside the cuff to activate motor nerve fibers running within the nearby superior laryngeal branch of the vagus. Activation of these muscles generated artifacts in the ENG recordings that may be mistaken for compound action potentials representing slowly conducting Aδ-, B-, and C-fibers. Significance: Our data resolve conflicting reports of the stimulation amplitudes required for C-fiber activation in large animal studies (>10 mA) and human studies (<250 µA). After removing muscle-generated artifacts, ENG signals with post-stimulus latencies consistent with Aδ- and B-fibers occurred in only a small subset of animals, and these signals had similar thresholds to those that caused bradycardia. By identifying specific neuroanatomical pathways that cause off-target effects and characterizing the stimulation dose-response curves for on- and off-target effects, we hope to guide interpretation and optimization of clinical VNS.

Functional vagotopy in the cervical vagus nerve of the domestic pig: implications for the study of vagus nerve stimulation.

OBJECTIVE: Given current clinical interest in vagus nerve stimulation (VNS), there are surprisingly few studies characterizing the anatomy of the vagus nerve in large animal models as it pertains to on-and off-target engagement of local fibers. We sought to address this gap by evaluating vagal anatomy in the pig, whose vagus nerve organization and size approximates the human vagus nerve. APPROACH: Here we combined microdissection, histology, and immunohistochemistry to provide data on key features across the cervical vagus nerve in a swine model, and compare our results to other animal models (mouse, rat, dog, non-human primate) and humans. MAIN RESULTS: In a swine model we quantified the nerve diameter, number and diameter of fascicles, and distance of fascicles from the epineural surface where stimulating electrodes are placed. We also characterized the relative locations of the superior and recurrent laryngeal branches of the vagus nerve that have been implicated in therapy limiting side effects with common electrode placement. We identified key variants across the cohort that may be important for VNS with respect to changing sympathetic/parasympathetic tone, such as cross-connections to the sympathetic trunk. We discovered that cell bodies of pseudo-unipolar cells aggregate together to form a very distinct grouping within the nodose ganglion. This distinct grouping gives rise to a larger number of smaller fascicles as one moves caudally down the vagus nerve. This often leads to a distinct bimodal organization, or 'vagotopy'. This vagotopy was supported by immunohistochemistry where approximately half of the fascicles were immunoreactive for choline acetyltransferase, and reactive fascicles were generally grouped in one half of the nerve. SIGNIFICANCE: The vagotopy observed via histology may be advantageous to exploit in design of electrodes/stimulation paradigms. We also placed our data in context of historic and recent histology spanning multiple models, thus providing a comprehensive resource to understand similarities and differences across species.

An Injectable Neural Stimulation Electrode Made from an In-Body Curing Polymer/Metal Composite.

Implanted neural stimulation and recording devices hold vast potential to treat a variety of neurological conditions, but the invasiveness, complexity, and cost of the implantation procedure greatly reduce access to an otherwise promising therapeutic approach. To address this need, a novel electrode that begins as an uncured, flowable prepolymer that can be injected around a neuroanatomical target to minimize surgical manipulation is developed. Referred to as the Injectrode, the electrode conforms to target structures forming an electrically conductive interface which is orders of magnitude less stiff than conventional neuromodulation electrodes. To validate the Injectrode, detailed electrochemical and microscopy characterization of its material properties is performed and the feasibility of using it to stimulate the nervous system electrically in rats and swine is validated. The silicone-metal-particle composite performs very similarly to pure wire of the same metal (silver) in all measures, including exhibiting a favorable cathodic charge storage capacity (CSCC ) and charge injection limits compared to the clinical LivaNova stimulation electrode and silver wire electrodes. By virtue of its simplicity, the Injectrode has the potential to be less invasive, more robust, and more cost-effective than traditional electrode designs, which could increase the adoption of neuromodulation therapies for existing and new indications.

Single-Dose Neoadjuvant AKT Pathway Inhibitor Reduces Growth of Hepatocellular Carcinoma after Laser Thermal Ablation in Small-Animal Model.

BackgroundLocal recurrence following thermal ablation of hepatocellular carcinoma (HCC) larger than 2-3 cm remains a challenging clinical problem. Prior studies suggest that phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)-dependent protein kinase B (AKT) signaling mediates HCC cell survival caused by moderate heat stress in vitro, but these findings need in vivo validation.PurposeTo test the hypothesis that neoadjuvant inhibition of PI3K/mTOR/AKT signaling reduces HCC tumor growth in vivo after laser ablation and to evaluate the effects of moderate heat stress on molecular signaling and cellular function in HCC cells in vitro.Materials and MethodsHCC tumor-bearing mice were randomized to neoadjuvant PI3K/mTOR inhibitor (BEZ235) or control groups followed by an intentional partial laser ablation or sham ablation; there were at least nine mice per group. Postablation tumor growth was monitored up to 7 days. Tumor volumes were compared for drug or ablation groups by using two-way analysis of variance. N1S1 HCC cells pretreated with BEZ235 or control and subjected to moderate heat stress (45°C for 10 minutes) or control (37°C for 10 minutes) were analyzed by using mass spectrometry. Protein interaction networks were derived from protein expression analysis software, and cellular function activation state (Z-score) and fold-change in AKT phosphorylation were calculated.ResultsThere was a 37%-75% reduction in HCC tumor volume by day 7 after ablation in the BEZ235 plus ablation group (713 mm3 ± 417) compared with vehicle plus sham (1559 mm3 ± 552), vehicle plus ablation (1041 mm3 ± 591), and BEZ235 plus sham (1108 mm3 ± 523) groups (P < .001, P = .04, and P = .005, respectively). PI3K/mTOR inhibition prevented moderate heat stress-induced AKT signaling (Z-score, -0.2; P < .001) and isoform-specific AKT phosphorylation compared with the vehicle plus heat stress group. PI3K/mTOR inhibition prevented moderate heat stress-induced global effects on HCC molecular signaling and cellular function, including decreased cell survival, growth, and proliferation (Z-score, -0.3 to -3.2; P < .001) and increased apoptosis and cell death (Z-score, 0.4-1.1; P < .001).ConclusionModerate heat stress induces PI3K/mTOR/AKT-dependent global effects on hepatocellular carcinoma (HCC) cell survival, function, and death. Neoadjuvant PI3K/mTOR/AKT inhibition reduces postablation HCC tumor growth.© RSNA, 2019Online supplemental material is available for this article.See also the editorial by White in this issue.

Functional Ultrasound Imaging of Spinal Cord Hemodynamic Responses to Epidural Electrical Stimulation: A Feasibility Study.

This study presents the first implementation of functional ultrasound (fUS) imaging of the spinal cord to monitor local hemodynamic response to epidural electrical spinal cord stimulation (SCS) on two small and large animal models. SCS has been successfully applied to control chronic refractory pain and recently was evolved to alleviate motor impairment in Parkinson's disease and after spinal cord injury. At present, however, the mechanisms underlying SCS remain unclear, and current methods for monitoring SCS are limited in their capacity to provide the required sensitivity and spatiotemporal resolutions to evaluate functional changes in response to SCS. fUS is an emerging technology that has recently shown promising results in monitoring a variety of neural activities associated with the brain. Here we demonstrated the feasibility of performing fUS on two animal models during SCS. We showed in vivo spinal cord hemodynamic responses measured by fUS evoked by different SCS parameters. We also demonstrated that fUS has a higher sensitivity in monitoring spinal cord response than electromyography. The high spatial and temporal resolutions of fUS were demonstrated by localized measurements of hemodynamic responses at different spinal cord segments, and by reliable tracking of spinal cord responses to patterned electrical stimulations, respectively. Finally, we proposed optimized fUS imaging and post-processing methods for spinal cord. These results support feasibility of fUS imaging of the spinal cord and could pave the way for future systematic studies to investigate spinal cord functional organization and the mechanisms of spinal cord neuromodulation in vivo.

Heat Stress and Thermal Ablation Induce Local Expression of Nerve Growth Factor Inducible (VGF) in Hepatocytes and Hepatocellular Carcinoma: Preclinical and Clinical Studies.

The purposes of this study were to test the hypothesis that heat stress and hepatic thermal ablation induce nerve growth factor inducible (VGF) and to determine intrahepatic versus systemic VGF expression induced by thermal ablation in vivo and in patients. Hepatocytes and HCC cells were subjected to moderate (45°C) or physiologic (37°C) heat stress for 10 min and assessed for VGF expression at 0-72 h post-heat stress (n ≥ 3 experiments). Orthotopic N1S1 HCC-bearing rats were randomized to sham or laser thermal ablation (3 W × 90 s), and liver/serum was harvested at 0-7 days postablation for analysis of VGF expression (n ≥ 6 per group). Serum was collected from patients undergoing thermal ablation for HCC (n = 16) at baseline, 3-6, and 18-24 h postablation and analyzed for VGF expression. Data were analyzed using ordinary or repeated-measures one-way analysis of variance and post hoc pairwise comparison with Dunnett's test. Moderate heat stress induced time-dependent VGF mRNA (3- to 15-fold; p < 0.04) and protein expression and secretion (3.1- to 3.3-fold; p < 0.05). Thermal ablation induced VGF expression at the hepatic ablation margin at 1 and 3 days postablation but not remote from the ablation zone or distant intrahepatic lobe. There was no detectable serum VGF following hepatic thermal ablation in rats and no increase in serum VGF following HCC thermal ablation in patients at 3-6 and 18-24 h postablation compared to baseline (0.71- and 0.63-fold; p = 0.27 and p = 0.16, respectively). Moderate heat stress induces expression and secretion of VGF in HCC cells and hepatocytes in vitro, and thermal ablation induces local intrahepatic but not distant intrahepatic or systemic VGF expression in vivo.

Heat Stress and Hepatic Laser Thermal Ablation Induce Hepatocellular Carcinoma Growth: Role of PI3K/mTOR/AKT Signaling.

Purpose To determine if heat stress and hepatic laser thermal ablation induce hepatocellular carcinoma (HCC) growth and to identify growth factors induced by heat stress. Materials and Methods Non-heat-stressed HCC cells were cocultured with HCC cells or hepatocytes that were heat stressed at 37°C (physiologic), 45°C (moderate), or 50°C (severe) for 10 minutes and proliferation monitored with bioluminescence imaging for up to 6 days after heat stress (three experiments). Rats bearing orthotopic N1S1 HCC were randomly assigned to undergo immediate sham or laser thermal (3 W for 60 or 90 seconds; hereafter, 3W×60s and 3W×90s, respectively) ablation of the median (local) or left (distant) hepatic lobe, and tumor growth was monitored with magnetic resonance imaging for up to 18 days after ablation (six or more rats per group). Experiments were repeated with rats randomly assigned to receive either the adjuvant phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor (NVP-BEZ235) or the vehicle control. Heat-stressed HCC cells and hepatocytes were analyzed by using microarray or quantitative real-time polymerase chain reaction analysis for growth factor expression (three or more experiments). Groups were compared by using one- or two-way analysis of variance, and post hoc pairwise comparison was performed with the Dunnett test. Results There were more non-heat-stressed HCC cells when cells were cocultured with cells subjected to moderate but not physiologic or severe heat stress (P < .001 for both). Local intrahepatic N1S1 tumors were larger at day 18 in the 3W×60s (mean, 3102 mm3 ± 463 [standard error]; P = .004) and 3W×90s (mean, 3538 mm3 ± 667; P < .001) groups than in the sham group (mean, 1363 mm3 ± 361) but not in distant intrahepatic tumors (P = .31). Adjuvant BEZ235 resulted in smaller N1S1 tumors in the BEZ235 and laser thermal ablation group than in the vehicle control and laser thermal ablation group (mean, 1731 mm3 ± 1457 vs 3844 mm3 ± 2400, P < .001). Moderate heat stress induced expression of growth factors in HCC cells and hepatocytes, including heparin-binding growth factor, fibroblast growth factor 21, and nerve growth factor (range, 2.9-66.9-fold; P < .05). Conclusion Moderate heat stress and laser thermal ablation induce hepatocellular carcinoma growth, which is prevented with adjuvant PI3K/mTOR/protein kinase B inhibition.

Heat stress induced, ligand-independent MET and EGFR signalling in hepatocellular carcinoma.

PURPOSE: The aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling. MATERIALS AND METHODS: MET+ and EGFR+ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control ± HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining. RESULTS: Heat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling. CONCLUSIONS: Heat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.

Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2-3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.

Blockade of CCR2 reduces macrophage influx and development of chronic renal damage in murine renovascular hypertension.

Renovascular hypertension (RVH) is a common cause of both cardiovascular and renal morbidity and mortality. In renal artery stenosis (RAS), atrophy in the stenotic kidney is associated with an influx of macrophages and other mononuclear cells. We tested the hypothesis that chemokine receptor 2 (CCR2) inhibition would reduce chronic renal injury by reducing macrophage influx in the stenotic kidney of mice with RAS. We employed a well-established murine model of RVH to define the relationship between macrophage infiltration and development of renal atrophy in the stenotic kidney. To determine the role of chemokine ligand 2 (CCL2)/CCR2 signaling in the development of renal atrophy, mice were treated with the CCR2 inhibitor RS-102895 at the time of RAS surgery and followed for 4 wk. Renal tubular epithelial cells expressed CCL2 by 3 days following surgery, a time at which no significant light microscopic alterations, including interstitial inflammation, were identified. Macrophage influx increased with time following surgery. At 4 wk, the development of severe renal atrophy was accompanied by an influx of inducible nitric oxide synthase (iNOS)+ and CD206+ macrophages that coexpressed F4/80, with a modest increase in macrophages coexpressing arginase 1 and F4/80. The CCR2 inhibitor RS-102895 attenuated renal atrophy and significantly reduced the number of dual-stained F4/80+ iNOS+ and F4/80+ CD206+ but not F4/80+ arginase 1+ macrophages. CCR2 inhibition reduces iNOS+ and CD206+ macrophage accumulation that coexpress F4/80 and renal atrophy in experimental renal artery stenosis. CCR2 blockade may provide a novel therapeutic approach to humans with RVH.

Combined effect of hyperfiltration and renin angiotensin system activation on development of chronic kidney disease in diabetic db/db mice.

BACKGROUND: Hypertension is a major risk factor for renal disease progression. However, the mechanisms by which hypertension aggravates the effects of diabetes on the kidney are incompletely understood. We tested the hypothesis that renovascular hypertension accelerates angiotensin-II-dependent kidney damage and inflammation in the db/db mouse, a model of type II diabetes. METHODS: Renovascular hypertension was established in db/db and wild-type control mice through unilateral renal artery stenosis (RAS); the non-stenotic contralateral kidneys evaluated 2, 4 and 6 weeks later. Angiotensin-II infusion (1000 ng/kg/min), unilateral nephrectomy, or both were also performed in db/db mice to discern the contributions of hypertension versus hyperfiltration to development of chronic renal injury in db/db mice with RAS. The effect of blood pressure reduction in db/db mice with RAS was assessed using angiotensin-receptor-blocker (ARB) or hydralazine treatment. RESULTS: Db/db mice with renovascular hypertension developed greater and more prolonged elevation of renin activity than all other groups studied. Stenotic kidneys of db/db mice developed progressive interstitial fibrosis, tubular atrophy, and interstitial inflammation. Contralateral kidneys of wild type mice with RAS showed minimal histopathologic abnormalities, whereas db/db mice with RAS developed severe diffuse mesangial sclerosis, interstitial fibrosis, tubular atrophy, and interstitial inflammation. Db/db mice with Angiotensin II-induced hypertension developed interstitial lesions and albuminuria but not mesangial matrix expansion, while nephrectomized db/db mice exhibited modest mesangial expansion and interstitial fibrosis, but not significant albuminuria. The combination of unilateral nephrectomy and angiotensin II infusion reproduced all the features of the injury albeit in a less severe manner. ARB and hydralazine were equally effective in attenuating the development of mesangial expansion in the contralateral kidneys of db/db mice with RAS. However, only ARB prevented elevation of urinary albumin/creatinine in db/db mice with RAS. CONCLUSION: Renovascular hypertension superimposed on diabetes exacerbates development of chronic renal disease in db/db mice at least in part through interaction with the renin-angiotensin system. Both ARB and hydralazine were equally effective in reducing systolic blood pressure and in preventing renal injury in the contralateral kidney of db/db mice with renal artery stenosis. ARB but not hydralazine prevented elevation of urinary albumin/creatinine in the db/db RAS model.

Anatomy of hepatic arteriolo-portal venular shunts evaluated by 3D micro-CT imaging.

The liver differs from other organs in that two vascular systems deliver its blood - the hepatic artery and the portal vein. However, how the two systems interact is not fully understood. We therefore studied the microvascular geometry of rat liver hepatic artery and portal vein injected with the contrast polymer Microfil(®). Intact isolated rat livers were imaged by micro-CT and anatomic evidence for hepatic arteriolo-portal venular shunts occurring between hepatic artery and portal vein branches was found. Simulations were performed to rule out the possibility of the observed shunts being artifacts resulting from image blurring. In addition, in the case of specimens where only the portal vein was injected, only the portal vein was opacified, whereas in hepatic artery injections, both the hepatic artery and portal vein were opacified. We conclude that mixing of the hepatic artery and portal vein blood can occur proximal to the sinusoidal level, and that the hepatic arteriolo-portal venular shunts may function as a one-way valve-like mechanism, allowing flow only from the hepatic artery to the portal vein (and not the other way around).

Methodological differences account for inconsistencies in reported free VEGF concentrations in pregnant rats.

Free vascular endothelial growth factor (VEGF) is undetectable in plasma during human pregnancy. However, studies examining pregnant rats have reported both low (8-29 pg/ml) and high (527-1,030 pg/ml) free VEGF. These discrepancies cast uncertainty over the use of rat models to study angiogenic factors in pregnancy and preeclampsia. This study investigates methodological factors that may explain these discrepancies. Plasma VEGF in nonpregnant, day 7 pregnant, and day 19 pregnant rats was measured using rat and mouse ELISAs (R&D Systems). The rat ELISA detected VEGF in plasma from nonpregnant rats but not in plasma from day 19 pregnant rats. The mouse ELISA detected higher VEGF concentrations than the rat ELISA in every sample tested. This discrepancy was greater in day 19 pregnant rats (median: 2,273 vs. 0 pg/ml) than in nonpregnant (97 vs. 20 pg/ml) and day 7 pregnant (66 vs. 2 pg/ml) rats. Recovery of recombinant rat VEGF (rrVEGF) spiked into plasma from nonpregnant and day 7 pregnant rats was high for the rat ELISA (82-105%) but low for the mouse ELISA (17-22%). The rat ELISA did not recover rrVEGF in plasma from day 19 pregnant rats, suggesting that this ELISA measures free VEGF. The use of the rat versus mouse ELISA likely explains the differences in reported VEGF concentrations in pregnant rats. While the rat ELISA appears to measure free VEGF, plasma concentrations in nonpregnant and pregnant rats are below the assay sensitivity limit. As most previous studies of pregnant rats used the mouse VEGF ELISA, these data should be interpreted cautiously.

Evolution of cardiac and renal impairment detected by high-field cardiovascular magnetic resonance in mice with renal artery stenosis.

BACKGROUND: Renal artery stenosis (RAS) promotes hypertension and cardiac dysfunction. The 2-kidney, 1-clip mouse model in many ways resembles RAS in humans and is amenable for genetic manipulation, but difficult to evaluate noninvasively. We hypothesized that cardiovascular magnetic resonance (CMR) is capable of detecting progressive cardiac and renal dysfunction in mice with RAS and monitoring the progression of the disease longitudinally. METHODS: RAS was induced at baseline in eighteen mice by constricting the renal artery. Nine additional animals served as normal controls. CMR scans (16.4 T) were performed in all mice one week before and 2 and 4 weeks after baseline. Renal volumes and hemodynamics were assessed using 3D fast imaging with steady-state precession and arterial spin labelling, and cardiac function using CMR cine. Renal hypoxia was investigated using blood oxygen-level dependent (BOLD) MR. RESULTS: Two weeks after surgery, mean arterial pressure was elevated in RAS mice. The stenotic kidney (STK) showed atrophy, while the contra-lateral kidney (CLK) showed hypertrophy. Renal blood flow (RBF) and cortical oxygenation level declined in the STK but remained unchanged in CLK. Moreover, cardiac end-diastolic and stroke volumes decreased and myocardial mass increased. At 4 weeks, STK RBF remained declined and the STK cortex and medulla showed development of hypoxia. Additionally, BOLD detected a mild hypoxia in CLK cortex. Cardiac end-diastolic and stroke volumes remained reduced and left ventricular hypertrophy worsened. Left ventricular filling velocities (E/A) indicated progression of cardiac dysfunction towards restrictive filling. CONCLUSIONS: CMR detected longitudinal progression of cardiac and renal dysfunction in 2K, 1C mice. These observations support the use of high-field CMR to obtain useful information regarding chronic cardiac and renal dysfunction in small animals.

Redox signaling is an early event in the pathogenesis of renovascular hypertension.

Activation of the renin-angiotensin-aldosterone system plays a critical role in the development of chronic renal damage in patients with renovascular hypertension. Although angiotensin II (Ang II) promotes oxidative stress, inflammation, and fibrosis, it is not known how these pathways intersect to produce chronic renal damage. We tested the hypothesis that renal parenchymal cells are subjected to oxidant stress early in the development of RVH and produce signals that promote influx of inflammatory cells, which may then propagate chronic renal injury. We established a reproducible murine model of RVH by placing a tetrafluoroethylene cuff on the right renal artery. Three days after cuff placement, renal tissue demonstrates no histologic abnormalities despite up regulation of both pro- and anti-oxidant genes. Mild renal atrophy was observed after seven days and was associated with induction of Tnfα and influx of CD3⁺ T cells and F4/80⁺ macrophages. By 28 days, kidneys developed severe renal atrophy with interstitial inflammation and fibrosis, despite normalization of plasma renin activity. Based on these considerations, we propose that renal parenchymal cells initiate a progressive cascade of events leading to oxidative stress, interstitial inflammation, renal fibrosis, and atrophy.

Non-invasive assessment of cardiac function in a mouse model of renovascular hypertension.

Hypertension continues to be a significant cause of morbidity and mortality, underscoring the need to better understand its early effects on the myocardium. The aim of this study is to determine the feasibility of in vivo longitudinal assessment of cardiac function, particularly diastolic function, in a mouse model of renovascular hypertension. Renovascular hypertension (RVH) was induced in 129S1/SvImJ male mice (n=9). To assess left ventricular (LV) systolic and diastolic function, M-mode echocardiography, pulsed-wave Doppler echocardiography and tissue Doppler imaging were performed at baseline, 2 and 4 weeks after the induction of renal artery stenosis. Myocardial tissue was collected to assess cellular morphology, fibrosis, extracellular matrix remodeling and inflammation ex vivo. RVH led to a significant increase in systolic blood pressure after 2 and 4 weeks (baseline: 99.26±1.09 mm Hg; 2 weeks: 140.90±7.64 mm Hg; 4 weeks: 147.52±5.91 mm Hg, P<0.05), resulting in a significant decrease in LV end-diastolic volume, associated with a significant elevation in ejection fraction and preserved cardiac output. Furthermore, the animals developed an abnormal diastolic function profile, with a shortening in the E velocity deceleration time as well as increases in the E/e' and the E/A ratio. The ex vivo analysis revealed a significant increase in myocyte size and deposition of extracellular matrix. Non-invasive high-resolution ultrasonography allowed assessment of the diastolic function profile in a small animal model of renovascular hypertension.