Repetitive Trans Spinal Magnetic Stimulation Improves Functional Recovery and Tissue Repair in Contusive and Penetrating Spinal Cord Injury Models in Rats.

Spinal cord injury (SCI) is an incurable condition in which the brain is disconnected partially or completely from the periphery. Mainly, SCIs are traumatic and are due to traffic, domestic or sport accidents. To date, SCIs are incurable and, most of the time, leave the patients with a permanent loss of sensitive and motor functions. Therefore, for several decades, researchers have tried to develop treatments to cure SCI. Among them, recently, our lab has demonstrated that, in mice, repetitive trans-spinal magnetic stimulation (rTSMS) can, after SCI, modulate the lesion scar and can induce functional locomotor recovery non-invasively. These results are promising; however, before we can translate them to humans, it is important to reproduce them in a more clinically relevant model. Indeed, SCIs do not lead to the same cellular events in mice and humans. In particular, SCIs in humans induce the formation of cystic cavities. That is why we propose here to validate the effects of rTSMS in a rat animal model in which SCI leads to the formation of cystic cavities after penetrating and contusive SCI. To do so, several techniques, including immunohistochemical, behavioral and MRI, were performed. Our results demonstrate that rTSMS, in both SCI models, modulates the lesion scar by decreasing the formation of cystic cavities and by improving axonal survival. Moreover, rTSMS, in both models, enhances functional locomotor recovery. Altogether, our study describes that rTSMS exerts positive effects after SCI in rats. This study is a further step towards the use of this treatment in humans.

Magnetic resonance colonography assessment of acute trinitrobenzene sulfonic acid colitis in pre-pubertal rats.

Pre-pubertal murine models of acute colitis are lacking. Magnetic resonance colonography (MRC) is a promising minimally invasive tool to assess colitis. We aimed to: 1/ Adapt a model of acute experimental colitis to pre-pubertal rats and determine whether MRC characteristics correlate with histological inflammation. 2/ Test this model by administering a diet supplemented in transforming growth factor ß2 to reverse inflammation. Twenty-four rats were randomized at weaning to one of 3 groups: Trinitrobenzene Sulfonic Acid (TNBS) group (n = 8) fed a standard diet, that received an intra-rectal 60 mg/kg dose of TNBS-ethanol; Control group (n = 8) fed standard diet, that received a dose of intra-rectal PBS; TNBS+MODULEN group (n = 8) that received a dose of TNBS and were exclusively fed MODULEN-IBD® after induction of colitis. One week after induction of colitis, rats were assessed by MRC, colon histopathology and inflammation markers (Interleukin 1ß, Tumor necrosis factor α, Nitric Oxide Synthase 2 and Cyclooxygenase 2). TNBS induced typical features of acute colitis on histopathology and MRC (increased colon wall thickness, increased colon intensity on T2-weighted images, target sign, ulcers). Treatment with MODULEN-IBD® did not reduce signs of colitis on MRC. Inflammatory marker expression did not differ among study groups.

Transient heart rate reduction improves acute decompensated heart failure-induced left ventricular and coronary dysfunction.

AIMS: Acute decompensated heart failure (ADHF), a live-threatening complication of heart failure (HF), associates a further decrease of the already by HF-impaired cardiac function with an increase in heart rate. We evaluated, using a new model of ADHF, whether heart rate reduction (HRR) opposes the acute decompensation-related aggravation of cardiovascular dysfunction. METHODS AND RESULTS: Cardiac output (echocardiography), cardiac tissue perfusion (magnetic resonance imaging), pulmonary wet weight, and in vitro coronary artery relaxation (Mulvany) were assessed 1 and 14 days after acute decompensation induced by salt-loading (1.8 g/kg, PO) in rats with well-established HF due to coronary ligation. HRR was induced by administration of the If current inhibitor S38844, 12 mg/kg PO twice daily for 2.5 days initiated 12 h or 6 days after salt-loading (early or delayed treatment, respectively). After 24 h, salt-loading resulted in acute decompensation, characterized by a reduction in cardiac output (HF: 130 ± 5 mL/min, ADHF: 105 ±  8 mL/min; P < 0.01), associated with a decreased myocardial perfusion (HF: 6.41 ± 0.53 mL/min/g, ADHF: 4.20 ± 0.11 mL/min/g; P < 0.01), a slight increase in pulmonary weight (HF: 1.68 ± 0.09 g, ADHF: 1.81 ± 0.15 g), and impaired coronary relaxation (HF: 55 ± 1% of pre-contraction at acetylcholine 4.5 10-5  M, ADHF: 27 ± 7 %; P < 0.01). Fourteen days after salt-loading, cardiac output only partially recovered (117 ± 5 mL/min; P < 0.05), while myocardial tissue perfusion (4.51 ± 0.44 mL/min; P < 0.01) and coronary relaxation (28 ± 4%; P < 0.01) remained impaired, but pulmonary weight further increased (2.06 ± 0.15 g, P < 0.05). Compared with untreated ADHF, HRR induced by S38844 improved cardiac output (125 ± 1 mL/min; P < 0.05), myocardial tissue perfusion (6.46 ± 0.42 mL/min/g; P < 0.01), and coronary relaxation (79 ± 2%; P < 0.01) as soon as 12 h after S38844 administration. These effects persisted beyond S38844 administration, illustrated by the improvements in cardiac output (130 ± 6 mL/min; P < 0.05), myocardial tissue perfusion (6.38 ± 0.48 mL/min/g; P < 0.01), and coronary relaxation (71 ± 4%; P < 0.01) at Day 14. S38844 did not modify pulmonary weight at Day 1 (1.78 ± 0.04 g) but tended to decrease pulmonary weight at Day 14 (1.80 ± 0.18 g). While delayed HRR induced by S38844 never improved cardiac function, early HRR rendered less prone to a second acute decompensation. CONCLUSIONS: In a model mimicking human ADHF, early, but not delayed, transient HRR induced by the If current inhibitor S38844 opposes acute decompensation by preventing the decompensated-related aggravation of cardiovascular dysfunction as well as the development of pulmonary congestion, and these protective effects persist beyond the transient treatment. Whether early transient HRR induced by If current inhibitors or other bradycardic agents, i.e. beta-blockers, exerts beneficial effects in human ADHF warrants further investigation.

A polymeric diet rich in transforming growth factor beta 2 does not reduce inflammation in chronic 2,4,6-trinitrobenzene sulfonic acid colitis in pre-pubertal rats.

BACKGROUND: Pediatric Crohn's disease is characterized by a higher incidence of complicated phenotypes. Murine models help to better understand the dynamic process of intestinal fibrosis and test therapeutic interventions. Pre-pubertal models are lacking. We aimed to adapt a model of chronic colitis to pre-pubertal rats and test if a polymeric diet rich in TGF-ß2 could reduce TNBS-induced intestinal inflammation and fibrosis. METHODS: Colitis was induced in 20 five-week-old Sprague-Dawley male rats by weekly rectal injections of increasing doses of TNBS (90 mg/kg, 140 mg/kg and 180 mg/kg) for 3 weeks, while 10 controls received phosphate-buffered saline. Rats were anesthetized using ketamine and chlorpromazine. After first administration of TNBS, 10 rats were fed exclusively MODULEN IBD® powder, while remaining rats were fed breeding chow. Colitis was assessed one week after last dose of TNBS by histopathology and magnetic resonance colonography (MRC). RESULTS: Histological inflammation and fibrosis scores were higher in TNBS group than controls (p < 0.05 for both). MRC showed increased colon wall thickness in TNBS group compared to controls (p < 0.01), and increased prevalence of strictures and target sign (p < 0.05). Colon expression of COL1A1, CTGF, α-SMA and COX-2 did not differ between TNBS rats and controls. TNBS colitis was not associated with growth failure. Treatment with MODULEN IBD® was associated with growth failure, increased colon weight/length ratio (p < 0.01), but did not affect histological scores or MRI characteristics. Colon expression of α-SMA was significantly lower in the MODULEN group versus controls (p = 0.005). CONCLUSION: Features of chronic colitis were confirmed in this model, based on MRC and histopathology. Treatment with MODULEN did not reverse inflammation or fibrosis.

Major hepatectomy decreased tumor growth in an experimental model of bilobar liver metastasis.

BACKGROUND/PURPOSE: Two-stage hepatectomy (TSH), is associated with a risk of drop-out due to tumoral progression following portal vein occlusion (PVO). We explored the impact of majorhepatectomy on tumor growth by objective radiological measures comparing to PVO and minor hepatectomy, using a model of bilobar colorectal liver metastasis (CLM). METHODS: CLM were induced in 48 BDIX rats by injection of DHDK12-cells. 7 days after cells injection, animals were distributed into 4 groups of equal number (n = 12): portal vein ligation (PVL), sham laparotomy (sham), minor (30%Phx) and major (70%Phx) hepatectomy. MR imaging was used for in vivo analysis of tumor implantation, growth and volumes. RESULTS: At POD10, tumour volumes were homogeneously distributed among the 4 groups. Lower TV were significantly observed after 70%Phx comparing to PVL at POD17 (0.63 ± 0.14cm3 vs 0.9 ± 0.16cm3, p = 0.008) and to the 3 others groups at POD24: 1.78 ± 0.38cm3 vs 3.2 ± 0.62cm3 (PVL, p = 0.019), 2.41 ± 0.74cm3 (Sham, p = 0.024) and 2.32 ± 0.59cm3 (30%PHx, p = 0.019). CONCLUSION: We confirmed in a reproducible model that contrary to PVO, a major hepatectomy decreases the growth of CLM in the remnant liver. This result leads to questioning the usual TSH and justifies exploring alternative strategies. The "major hepatectomy first-approach" should be an option to be evaluated.

Contribution of genotoxic anticancer treatments to the development of multiple primary tumours in the context of germline TP53 mutations.

INTRODUCTION: Li-Fraumeni syndrome (LFS), due to TP53 germline mutations, is characterised by a remarkably high incidence of multiple primary cancers (MPCs), and the key role of p53 in response to DNA damage questions the contribution of anticancer treatments to MPCs development. MATERIALS AND METHODS: We first evaluated genotoxicity of X-rays and different classes of conventional chemotherapies, thanks to genotoxicity assays, based on the measurement of transcriptional response to DNA damage and performed in murine splenocytes, either exposed ex vivo or extracted from exposed mice. We then exposed a total of 208 Trp53Δ/Δ, wt/Δ or wt/wt mice to clinical doses of X-rays or genotoxic or non-genotoxic chemotherapies. Tumour development was monitored using whole-body magnetic resonance imaging and pathological examination at death. RESULTS: X-rays and conventional chemotherapies, except mitotic spindle poisons, were found to be genotoxic in both p53 genotoxicity assays. Exposition to X-rays and the topoisomerase inhibitor etoposide, analysed as genotoxic anticancer treatment, drastically increase the tumour development risk in Trp53Δ/Δ and wt/Δ mice (hazard ration [HR] = 4.4, 95% confidence interval [CI] [2.2-8.8], p < 0.001*** and HR = 4.7, 95% CI [2.4-9.3], p < 0.001***, respectively). In contrast, exposure to the non-genotoxic mitotic spindle poison, docetaxel, had no impact on tumour development. CONCLUSIONS: This study shows that radiotherapy and genotoxic chemotherapies significantly increase the risk of tumour development in a LFS mice model. These results strongly support the contribution of genotoxic anticancer treatments to MPC development in LFS patients. Therefore, to reduce the risk of MPCs in germline TP53 mutation carriers, radiotherapy should be avoided whenever possible, surgical treatment prioritised, and non-genotoxic treatments considered.

Selective Vascular Endothelial Protection Reduces Cardiac Dysfunction in Chronic Heart Failure.

BACKGROUND: Chronic heart failure (CHF) induces endothelial dysfunction in part because of decreased nitric oxide (NO(·)) production, but the direct link between endothelial dysfunction and aggravation of CHF is not directly established. We previously reported that increased NO production via inhibition of protein tyrosine phosphatase 1B (PTP1B) is associated with reduced cardiac dysfunction in CHF. Investigation of the role of endothelial PTP1B in these effects may provide direct evidence of the link between endothelial dysfunction and CHF. METHODS AND RESULTS: Endothelial deletion of PTP1B was obtained by crossing LoxP-PTP1B with Tie2-Cre mice. CHF was assessed 4 months after myocardial infarction. In some experiments, to exclude gene extinction in hematopoietic cells, Tie2-Cre/LoxP-PTP1B mice were lethally irradiated and reconstituted with bone marrow from wild-type mice, to obtain mouse with endothelial-specific deletion of PTP1B. Vascular function evaluated ex vivo in mesenteric arteries showed that in wild-type mice, CHF markedly impaired NO-dependent flow-mediated dilatation. CHF-induced endothelial dysfunction was less marked in endoPTP1B(-/-) mice, suggesting restored NO production. Echocardiographic, hemodynamic, and histological evaluations demonstrated that the selectively improved endothelial function was associated with reduced left ventricular dysfunction and remodeling, as well as increased survival, in the absence of signs of stimulated angiogenesis or increased cardiac perfusion. CONCLUSIONS: Prevention of endothelial dysfunction, by endothelial PTP1B deficiency, is sufficient to reduce cardiac dysfunction post myocardial infarction. Our results provide for the first time a direct demonstration that endothelial protection per se reduces CHF and further suggest a causal role for endothelial dysfunction in CHF development.

Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction.

BACKGROUND: The lymphatic system regulates interstitial tissue fluid balance, and lymphatic malfunction causes edema. The heart has an extensive lymphatic network displaying a dynamic range of lymph flow in physiology. Myocardial edema occurs in many cardiovascular diseases, eg, myocardial infarction (MI) and chronic heart failure, suggesting that cardiac lymphatic transport may be insufficient in pathology. Here, we investigate in rats the impact of MI and subsequent chronic heart failure on the cardiac lymphatic network. Further, we evaluate for the first time the functional effects of selective therapeutic stimulation of cardiac lymphangiogenesis post-MI. METHODS AND RESULTS: We investigated cardiac lymphatic structure and function in rats with MI induced by either temporary occlusion (n=160) or permanent ligation (n=100) of the left coronary artery. Although MI induced robust, intramyocardial capillary lymphangiogenesis, adverse remodeling of epicardial precollector and collector lymphatics occurred, leading to reduced cardiac lymphatic transport capacity. Consequently, myocardial edema persisted for several months post-MI, extending from the infarct to noninfarcted myocardium. Intramyocardial-targeted delivery of the vascular endothelial growth factor receptor 3-selective designer protein VEGF-CC152S, using albumin-alginate microparticles, accelerated cardiac lymphangiogenesis in a dose-dependent manner and limited precollector remodeling post-MI. As a result, myocardial fluid balance was improved, and cardiac inflammation, fibrosis, and dysfunction were attenuated. CONCLUSIONS: We show that, despite the endogenous cardiac lymphangiogenic response post-MI, the remodeling and dysfunction of collecting ducts contribute to the development of chronic myocardial edema and inflammation-aggravating cardiac fibrosis and dysfunction. Moreover, our data reveal that therapeutic lymphangiogenesis may be a promising new approach for the treatment of cardiovascular diseases.

Magnetic resonance colonography for fibrosis assessment in rats with chronic colitis.

BACKGROUND: Magnetic resonance colonography (MRC) has been developed to assess inflammatory bowel diseases. We aimed to assess the feasibility of MRC in rats with TNBS-induced chronic colitis and to confront imaging results with fibrosis and stenosing features of the model. MATERIALS AND METHODS: Chronic colitis was induced in 12 rats by weekly intra-rectal injection of increasing doses of TNBS for 6 weeks, while 8 control rats received the vehicle. At week 7, MRC was performed. Fibrosis scores were assessed and fibrosis mediators measured. RESULTS: Chronic colitis was associated with significant body weight loss (p<0.0001) and higher colon weight/length compared to controls (p = 0.0004). Fibrosis mediators and histological scores were significantly higher in rats with TNBS than in controls: α-SMA expression (0.9 versus 0.61, p = 0.0311) and fibrosis score (p = 0.0308). Colon wall thickness was higher in rats with TNBS than in controls: maximal thickness (2.38 versus 0.74 mm, p<0.0001) and minimal thickness (1.33 versus 0.48 mm, p<0.0001). Wall signal intensity on T2w images was higher in rats with TNBS than in controls (9040 versus 6192, p = 0.0101) and correlated with fibrosis score (r = 0.5214; p = 0.04). Luminal narrowing was higher in rats with TNBS (50.08 versus 10.33%, p<0.0001) and correlated with α-SMA expression (r = 0.5618; p = 0.01). Stenosis was observed in 7/9 rats with TNBS and in no controls (p = 0.0053). CONCLUSIONS: MRC is feasible and easily distinguishes rats with colitis from controls. MRC signs correlated with fibrosis parameters. MRC evaluation may be part of a new anti-fibrosis drug assessment in experimental models of chronic colitis.

Magnetic resonance colonography in rats with TNBS-induced colitis: a feasibility and validation study.

BACKGROUND: Magnetic resonance colonography (MRC) has been recently developed to assess bowel inflammation in inflammatory bowel disease (IBD) patients. Evaluating animal models of inflammation with MRC may be important in new drug-screening processes. The aim of this study was to assess the feasibility of MRC in colitic rats and confront it with model characteristics. METHODS: Colitis was induced by rectal injection of trinitrobenzene-sulfonic acid (TNBS) in 13 rats while six rats received the vehicle. MRC was performed at day 2. Colon inflammation and production of inflammatory mediators were evaluated. Image quality was assessed by wall and motion artifacts. MRC criteria were bowel wall thickness, wall signal intensity on T2-weighted (T2w) and T1w images, the appearance of a target sign pattern, and irregular patterns of mucosal surface. RESULTS: MRC quality was good or excellent in 16/21 examinations with no difference between groups. Colitis rats were significantly different from controls in terms of wall thickness (P = 0.004), the appearance of a target sign pattern (P = 0.02), irregular patterns of mucosal surface (P = 0.01), and hyperintensity on T1w images (P = 0.03). All MRC criteria except maximal bowel wall thickness were associated with colon weight:length ratio and inflammatory biomarkers (all P < 0.05). Minimal bowel wall thickness and wall signal intensity on T2w images were associated with histological score (P < 0.05). CONCLUSIONS: MRC is feasible and reliable in rats with TNBS-induced colitis. MRC criteria including colon wall thickness, wall signal intensity on T2w images, hyperintensity in T1w sequence, and the appearance of a target sign pattern may be potential targets for new IBD drugs.

Soluble epoxide hydrolase inhibition improves myocardial perfusion and function in experimental heart failure.

The study addressed the hypothesis that soluble epoxide hydrolase (sEH) inhibition, which increases cardiovascular protective epoxyeicosatrienoic acids (EETs), exerts beneficial effects in an established chronic heart failure (CHF) model. In CHF rats, left ventricular (LV) function, perfusion and remodeling were assessed using MRI and invasive hemodynamics after 42-day (starting 8 days after coronary ligation) and delayed 3-day (starting 47 days after coronary ligation) treatments with the sEH inhibitor AUDA (twice 0.25 mg/day). Delayed 3-day and 42-day AUDA increased plasma EETs demonstrating the effective inhibition of sEH. Delayed 3-day and 42-day AUDA enhanced cardiac output without change in arterial pressure, thus reducing total peripheral resistance. Both treatment periods increased the slope of the LV end-systolic pressure-volume relation, but only 42-day AUDA decreased LV end-diastolic pressure, relaxation constant Tau and the slope of the LV end-diastolic pressure-volume relation, associated with a reduced LV diastolic volume and collagen density. Delayed 3-day and, to a larger extent, 42-day AUDA increased LV perfusion associated with a decreased LV hypoxia-inducible factor-1alpha. Both treatment periods decreased reactive oxygen species level and increased reduced-oxidized glutathione ratio. Finally, MSPPOH, an inhibitor of the EET-synthesizing enzyme cytochrome epoxygenases, abolished the beneficial effects of 3-day AUDA on LV function and perfusion. Augmentation of EET availability by pharmacological inhibition of sEH increases LV diastolic and systolic functions in established CHF. This notably results from short-term processes, i.e. increased LV perfusion, reduced LV oxidative stress and peripheral vasodilatation, but also from long-term effects, i.e. reduced LV remodeling.

Arteriogenic therapy by intramyocardial sustained delivery of a novel growth factor combination prevents chronic heart failure.

BACKGROUND: Therapeutic angiogenesis is a promising approach for the treatment of cardiovascular diseases, including myocardial infarction and chronic heart failure. We aimed to improve proangiogenic therapies by identifying novel arteriogenic growth factor combinations, developing injectable delivery systems for spatiotemporally controlled growth factor release, and evaluating functional consequences of targeted intramyocardial growth factor delivery in chronic heart failure. METHODS AND RESULTS: First, we observed that fibroblast growth factor and hepatocyte growth factor synergistically stimulate vascular cell migration and proliferation in vitro. Using 2 in vivo angiogenesis assays (n=5 mice per group), we found that the growth factor combination results in a more potent and durable angiogenic response than either growth factor used alone. Furthermore, we determined that the molecular mechanisms involve potentiation of Akt and mitogen-activated protein kinase signal transduction pathways, as well as upregulation of angiogenic growth factor receptors. Next, we developed crosslinked albumin-alginate microcapsules that sequentially release fibroblast growth factor-2 and hepatocyte growth factor. Finally, in a rat model of chronic heart failure induced by coronary ligation (n=14 to 15 rats per group), we found that intramyocardial slow release of fibroblast growth factor-2 with hepatocyte growth factor potently stimulates angiogenesis and arteriogenesis and prevents cardiac hypertrophy and fibrosis, as determined by immunohistochemistry, leading to improved cardiac perfusion after 3 months, as shown by magnetic resonance imaging. These multiple beneficial effects resulted in reduced adverse cardiac remodeling and improved left ventricular function, as revealed by echocardiography. CONCLUSION: Our data showing the selective advantage of using fibroblast growth factor-2 together with hepatocyte growth factor suggest that this growth factor combination may constitute an efficient novel treatment for chronic heart failure.