Inflammation triggers ILC3 patrolling of the intestinal barrier.

An orchestrated cellular network, including adaptive lymphocytes and group 3 innate lymphoid cells (ILC3s), maintains intestinal barrier integrity and homeostasis. T cells can monitor environmental insults through constitutive circulation, scanning tissues and forming immunological contacts, a process named immunosurveillance. In contrast, the dynamics of intestinal ILC3s are unknown. Using intravital imaging, we observed that villus ILC3s were largely immotile at steady state but acquired migratory 'patrolling' attributes and enhanced cytokine expression in response to inflammation. We showed that T cells, the chemokine CCL25 and bacterial ligands regulated intestinal ILC3 behavior and that loss of patrolling behavior by interleukin-22 (IL-22)-producing ILC3s altered the intestinal barrier through increased epithelial cell death. Collectively, we identified notable differences between the behavior of ILC3s and T cells, with a prominent adaptation of intestinal ILC3s toward mucosal immunosurveillance after inflammation.

Dichotomous metabolic networks govern human ILC2 proliferation and function.

Group 2 innate lymphoid cells (ILC2s) represent innate homologs of type 2 helper T cells (TH2) that participate in immune defense and tissue homeostasis through production of type 2 cytokines. While T lymphocytes metabolically adapt to microenvironmental changes, knowledge of human ILC2 metabolism is limited, and its key regulators are unknown. Here, we show that circulating 'naive' ILC2s have an unexpected metabolic profile with a higher level of oxidative phosphorylation (OXPHOS) than natural killer (NK) cells. Accordingly, ILC2s are severely reduced in individuals with mitochondrial disease (MD) and impaired OXPHOS. Metabolomic and nutrient receptor analysis revealed ILC2 uptake of amino acids to sustain OXPHOS at steady state. Following activation with interleukin-33 (IL-33), ILC2s became highly proliferative, relying on glycolysis and mammalian target of rapamycin (mTOR) to produce IL-13 while continuing to fuel OXPHOS with amino acids to maintain cellular fitness and proliferation. Our results suggest that proliferation and function are metabolically uncoupled in human ILC2s, offering new strategies to target ILC2s in disease settings.

A human immune system (HIS) mouse model that dissociates roles for mouse and human FcR+ cells during antibody-mediated immune responses.

Human immune system (HIS) mice provide a model to study human immune responses in vivo. Currently available HIS mouse models may harbor mouse Fc Receptor (FcR)-expressing cells that exert potent effector functions following administration of human Ig. Previous studies showed that the ablation of the murine FcR gamma chain (FcR-γ) results in loss of antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vivo. We created a new FcR-γ-deficient HIS mouse model to compare host (mouse) versus graft (human) effects underlying antibody-mediated immune responses in vivo. FcR-γ-deficient HIS recipients lack expression and function of mouse activating FcRs and can be stably and robustly reconstituted with human immune cells. By screening blood B-cell depletion by rituximab Ig variants, we found that human FcγRs-mediated IgG1 effects, whereas mouse activating FcγRs were dominant in IgG4 effects. Complement played a role as an IgG1 variant (IgG1 K322A) lacking complement binding activity was largely ineffective. Finally, we provide evidence that FcγRIIIA on human NK cells could mediate complement-independent B-cell depletion by IgG1 K322A. We anticipate that our FcR-γ-deficient HIS model will help clarify mechanisms of action of exogenous administered human antibodies in vivo.

Host genetic control of natural killer cell diversity revealed in the Collaborative Cross.

Natural killer (NK) cells are innate effectors armed with cytotoxic and cytokine-secreting capacities whose spontaneous antitumor activity is key to numerous immunotherapeutic strategies. However, current mouse models fail to mirror the extensive immune system variation that exists in the human population which may impact on NK cell-based therapies. We performed a comprehensive profiling of NK cells in the Collaborative Cross (CC), a collection of novel recombinant inbred mouse strains whose genetic diversity matches that of humans, thereby providing a unique and highly diverse small animal model for the study of immune variation. We demonstrate that NK cells from CC strains displayed a breadth of phenotypic and functional variation reminiscent of that reported for humans with regards to cell numbers, key marker expression, and functional capacities. We took advantage of the vast genetic diversity of the CC and identified nine genomic loci through quantitative trait locus mapping driving these phenotypic variations. SNP haplotype patterns and variant effect analyses identified candidate genes associated with lung NK cell numbers, frequencies of CD94+ NK cells, and expression levels of NKp46. Thus, we demonstrate that the CC represents an outstanding resource to study NK cell diversity and its regulation by host genetics.

State-of-the-art therapies for Rett syndrome.

Rett syndrome (RTT) is an X-linked neurogenetic disorder caused by mutations of the MECP2 (methyl-CpG-binding protein 2) gene. Over two decades of work established MeCP2 as a protein with pivotal roles in the regulation of the epigenome, neuronal physiology, synaptic maintenance, and behaviour. Given the genetic aetiology of RTT and the proof of concept of its reversal in a mouse model, considerable efforts have been made to design therapeutic approaches to re-express MeCP2. By being at the forefront of the development of innovative gene therapies, research on RTT is of paramount importance for the treatment of monogenic neurological diseases. Here we discuss the recent advances and challenges of promising genetic strategies for the treatment of RTT including gene replacement therapies, gene/RNA editing strategies, and reactivation of the silenced X chromosome. WHAT THIS PAPER ADDS: Recent advances shed light on the promises of gene replacement therapy with new vectors designed to control the levels of MeCP2 expression. New developments in DNA/RNA editing approaches or reactivation of the silenced X chromosome open the possibility to re-express the native MeCP2 locus at endogenous levels. Current strategies still face limitations in transduction efficiency and future work is needed to improve brain delivery.

A knock-in mouse model for KCNQ2-related epileptic encephalopathy displays spontaneous generalized seizures and cognitive impairment.

OBJECTIVE: Early onset epileptic encephalopathy with suppression-burst is one of the most severe epilepsy phenotypes in human patients. A significant proportion of cases have a genetic origin, and the most frequently mutated gene is KCNQ2, encoding Kv7.2, a voltage-dependent potassium channel subunit, leading to so-called KCNQ2-related epileptic encephalopathy (KCNQ2-REE). To study the pathophysiology of KCNQ2-REE in detail and to provide a relevant preclinical model, we generated and described a knock-in mouse model carrying the recurrent p.(Thr274Met) variant. METHODS: We introduced the p.(Thr274Met) variant by homologous recombination in embryonic stem cells, injected into C57Bl/6N blastocysts and implanted in pseudopregnant mice. Mice were then bred with 129Sv Cre-deleter to generate heterozygous mice carrying the p.(Thr274Met), and animals were maintained on the 129Sv genetic background. We studied the development of this new model and performed in vivo electroencephalographic (EEG) recordings, neuroanatomical studies at different time points, and multiple behavioral tests. RESULTS: The Kcnq2Thr274Met/+ mice are viable and display generalized spontaneous seizures first observed between postnatal day 20 (P20) and P30. In vivo EEG recordings show that the paroxysmal events observed macroscopically are epileptic seizures. The brain of the Kcnq2Thr274Met/+ animals does not display major structural defects, similar to humans, and their body weight is normal. Kcnq2Thr274Met/+ mice have a reduced life span, with a peak of unexpected death occurring for 25% of the animals by 3 months of age. Epileptic seizures were generally not observed when animals grew older. Behavioral characterization reveals important deficits in spatial learning and memory in adults but no gross abnormality during early neurosensory development. SIGNIFICANCE: Taken together, our results indicate that we have generated a relevant model to study the pathophysiology of KCNQ2-related epileptic encephalopathy and perform preclinical research for that devastating and currently intractable disease.

Phenotypic and Functional Plasticity of Murine Intestinal NKp46+ Group 3 Innate Lymphoid Cells.

Group 3 innate lymphoid cells (ILC3) actively participate in mucosal defense and homeostasis through prompt secretion of IL-17A, IL-22, and IFN-γ. Reports identify two ILC3 lineages: a CCR6(+)T-bet(-) subset that appears early in embryonic development and promotes lymphoid organogenesis and a CCR6(-)T-bet(+) subset that emerges after microbial colonization and harbors NKp46(+) ILC3. We demonstrate that NKp46 expression in the ILC3 subset is highly unstable. Cell fate mapping using Ncr1(CreGFP) × Rosa26(RFP) mice revealed the existence of an intestinal RFP(+) ILC3 subset (Ncr1(FM)) lacking NKp46 expression at the transcript and protein levels. Ncr1(FM) ILC3 produced more IL-22 and were distinguishable from NKp46(+) ILC3 by differential CD117, CD49a, DNAX accessory molecule-1, and, surprisingly, CCR6 expression. Ncr1(FM) ILC3 emerged after birth and persisted in adult mice following broad-spectrum antibiotic treatment. These results identify an unexpected phenotypic instability within NKp46(+) ILC3 that suggests a major role for environmental signals in tuning ILC3 functional plasticity.

Biocompatibility of Four Common Orthopedic Biomaterials Following a High-Salt Diet: An In Vivo Study.

Nowadays, salt consumption appears to be drastically above the recommended level in industrialized countries. The health consequences of this overconsumption are heavy since high-salt intake induces cardiovascular disease, kidney dysfunction, and stroke. Moreover, harmful interaction may also occur with orthopaedic devices because overconsumption of salt reinforces the corrosive aspect of biological tissues and favors bone resorption process. In the present study, we aimed to assess the in vivo effect of three weeks of a high-salt diet, associated (or not) with two weeks of the neuro-myoelectrostimulation (NMES) rehabilitation program on the biocompatibility of four biomaterials used in the manufacture of arthroplasty implants. Thus, two non-metallic (PEEK and Al2O3) and two metallic (Ti6Al4V and CrCo) compounds were implanted in the rat tibial crest, and the implant-to-bone adhesion and cell viability of two surrounded muscles, the Flexor Digitorum (FD) and Tibialis Anterior (TA), were assessed at the end of the experiment. Results indicated lower adhesion strength for the PEEK implant compared to other biomaterials. An effect of NMES and a high-salt diet was only identified for Al2O3 and Ti6Al4V implants, respectively. Moreover, compared to a normal diet, a high-salt diet induced a higher number of dead cells on both muscles for all biomaterials, which was further increased for PEEK, Al2O3, and CrCo materials with NMES application. Finally, except for Ti6Al4V, NMES induced a higher number of dead cells in the directly stimulated muscle (FD) compared to the indirectly stimulated one (TA). This in vivo experiment highlights the potential harmful effect of a high-salt diet for people who have undergone arthroplasty, and a rehabilitation program based on NMES.

Conditional ablation of NKp46+ cells using a novel Ncr1(greenCre) mouse strain: NK cells are essential for protection against pulmonary B16 metastases.

To study gene functions specifically in NKp46+ cells we developed novel Cre mice allowing for conditional gene targeting in cells expressing Ncr1 (encoding NKp46). We generated transgenic Ncr1(greenCre) mice carrying an EGFPcre fusion under the control of a proximal Ncr1 promoter that faithfully directed EGFPcre expression to NKp46+ cells from lymphoid and nonlymphoid tissues. This approach allowed for direct detection of Cre-expressing NKp46+ cells via their GFP signature by flow cytometry and histology. Cre was functional as evidenced by the NKp46+ cell-specific expression of RFP in Ncr1(greenCre) Rosa-dtRFP reporter mice. We generated Ncr1(greenCre) Il2rg(fl/fl) mice that lack NKp46+ cells in an otherwise intact hematopoietic environment. Il2rg encodes the common gamma chain (γc ), which is an essential receptor subunit for cytokines (IL-2, -4, -7, -9, -15, and -21) that stimulate lymphocyte development and function. In Ncr1(greenCre) Il2rg(fl/fl) mice, NK cells are severely reduced and the few remaining NKp46+ cells escaping γc deletion failed to express GFP. Using this new NK-cell-deficient model, we demonstrate that the homeostasis of NKp46+ cells from all tissues (including the recently described intraepithelial ILC1 subset) requires Il2rg. Finally, Ncr1(greenCre) Il2rg(fl/fl) mice are unable to reject B16 lung metastases demonstrating the essential role of NKp46+ cells in antimelanoma immune responses.

Anoctamin 1 dysregulation alters bronchial epithelial repair in cystic fibrosis.

Cystic fibrosis (CF) airway epithelium is constantly subjected to injury events due to chronic infection and inflammation. Moreover, abnormalities in CF airway epithelium repair have been described and contribute to the lung function decline seen in CF patients. In the last past years, it has been proposed that anoctamin 1 (ANO1), a Ca(2+)-activated Cl(-) channel, might offset the CFTR deficiency but this protein has not been characterized in CF airways. Interestingly, recent evidence indicates a role for ANO1 in cell proliferation and tumor growth. Our aims were to study non-CF and CF bronchial epithelial repair and to determine whether ANO1 is involved in airway epithelial repair. Here, we showed, with human bronchial epithelial cell lines and primary cells, that both cell proliferation and migration during epithelial repair are delayed in CF compared to non-CF cells. We then demonstrated that ANO1 Cl(-) channel activity was significantly decreased in CF versus non-CF cells. To explain this decreased Cl(-) channel activity in CF context, we compared ANO1 expression in non-CF vs. CF bronchial epithelial cell lines and primary cells, in lung explants from wild-type vs. F508del mice and non-CF vs. CF patients. In all these models, ANO1 expression was markedly lower in CF compared to non-CF. Finally, we established that ANO1 inhibition or overexpression was associated respectively with decreases and increases in cell proliferation and migration. In summary, our study demonstrates involvement of ANO1 decreased activity and expression in abnormal CF airway epithelial repair and suggests that ANO1 correction may improve this process.

Ultrasound-induced seizures in a mouse model of KCNQ2-NEO-DEE.

PURPOSE: KCNQ2 neonatal developmental and epileptic encephalopathy (NEO-DEE) is characterized by intractable seizures accompanied by an abnormal neurodevelopment. In a mouse model of NEO-DEE carrying the p.(Thr274Met) variant of Kcnq2, spontaneous generalized seizures occur unexpectedly preventing controlled studies and highlighting the necessity for a customized setup to trigger seizures on demand. We aimed to obtain a stable and objective read-out to control the efficacy of new antiepileptic drugs or to test seizure susceptibility. We developed a protocol to trigger ultrasound-induced seizures (UIS) on demand in this model. METHODS: We tested the ability of our protocol to induce seizures at four developmental stages in the Kcnq2p.(Thr274Met/+) mouse model. We mapped the activated brain regions using c-fos protein labeling 2 h after seizure induction. RESULTS: We show that the UIS have the same phenotypic expression and the same severity as spontaneous generalized seizures (SGS) in the Kcnq2-NEO-DEE mouse model. The developmental period during which mice exhibit SGS corresponds to the period during which Kcnq2p.(Thr274Met/+) mice are the most susceptible to US. C-fos labeling reveals a subset of 6 brain regions activated 2 h after the induction of the seizure. The same regions were identified in the context of seizure induction in other rodent models. CONCLUSION: This study provides a non-invasive and easy to use method to induce seizures in a Kcnq2-NEO-DEE mouse model and documents early neuronal activation in specific brain regions. This method can be used to test the efficacy of new antiepileptic approaches for this intractable form of genetic epilepsy.

Notch, RORC and IL-23 signals cooperate to promote multi-lineage human innate lymphoid cell differentiation.

Innate lymphoid cells (ILCs) include cytotoxic natural killer cells and distinct groups of cytokine-producing innate helper cells which participate in immune defense and promote tissue homeostasis. Circulating human ILC precursors (ILCP) able to generate all canonical ILC subsets via multi-potent or uni-potent intermediates according to our previous work. Here we show potential cooperative roles for the Notch and IL-23 signaling pathways for human ILC differentiation from blood ILCP using single cell cloning analyses and validate these findings in patient samples with rare genetic deficiencies in IL12RB1 and RORC. Mechanistically, Notch signaling promotes upregulation of the transcription factor RORC, enabling acquisition of Group 1 (IFN-γ) and Group 3 (IL-17A, IL-22) effector functions in multi-potent and uni-potent ILCP. Interfering with RORC or signaling through its target IL-23R compromises ILC3 effector functions but also generally suppresses ILC production from multi-potent ILCP. Our results identify a Notch->RORC- > IL-23R pathway which operates during human ILC differentiation. These observations may help guide protocols to expand functional ILC subsets in vitro with an aim towards novel ILC therapies for human disease.

Reduced expression of Tis7/IFRD1 protein in murine and human cystic fibrosis airway epithelial cell models homozygous for the F508del-CFTR mutation.

12-O-tetradecanoyl phorbol-13-acetate-induced sequence 7/interferon related development regulator 1 (Tis7/IFRD1) has been recently identified as a modifier gene in lung inflammatory disease severity in patients with cystic fibrosis (CF), based upon its capacity to regulate inflammatory activities in neutrophils. In CF patients, the F508del mutation in the Cftr gene encoding a chloride channel, the CF transmembrane conductance regulator (CFTR) in airway epithelial cells results in an exaggerated inflammatory response of these cells. At present, it is unknown whether the Tis7/IFRD1 gene product is expressed in airway epithelial cells. We therefore investigated the possibility there is an intrinsic alteration in Tis7/IFRD1 protein level in cells lacking CFTR function in tracheal homogenates of F508del-CFTR mice and in a F508del-CFTR human bronchial epithelial cell line (CFBE41o(-) cells). When Tis7/IFRD1 protein was detectable, trachea from F508del-CFTR mice showed a reduction in the level of Tis7/IFRD1 protein compared to wild-type control littermates. A significant reduction of IFRD1 protein level was found in CFBE41o(-) cells compared to normal bronchial epithelial cells 16HBE14o(-). Surprisingly, messenger RNA level of IFRD1 in CFBE41o(-) cells was found elevated. Treating CFBE41o(-) cells with the antioxidant glutathione rescued the IFRD1 protein level closer to control level and also reduced the pro-inflammatory cytokine IL-8 release. This work provides evidence for the first time of reduced level of IFRD1 protein in murine and human F508del-CFTR airway epithelial cell models, possibly mediated in response to oxidative stress which might contribute to the exaggerated inflammatory airway response observed in CF patients homozygous for the F508del mutation.

Combination of myostatin pathway interference and dystrophin rescue enhances tetanic and specific force in dystrophic mdx mice.

Duchenne muscular dystrophy is characterized by muscular atrophy, fibrosis, and fat accumulation. Several groups have demonstrated that in the mdx mouse, the exon-skipping strategy can restore a quasi-dystrophin in almost 100% of the muscle fibers. On the other hand, inhibition of the myostatin pathway in adult mice has been described to enhance muscle growth and improve muscle force. Our aim was to combine these two strategies to evaluate a possible additive effect. We have chosen to inhibit the myostatin pathway using the technique of RNA interference directed against the myostatin receptor AcvRIIb mRNA (sh-AcvRIIb). The restoration of a quasi-dystrophin was mediated by the vectorized U7 exon-skipping technique (U7-DYS). Adeno-associated vectors carrying either the sh-AcvrIIb construct alone, the U7-DYS construct alone, or a combination of both constructs were injected in the tibialis anterior (TA) muscle of dystrophic mdx mice. We show that even if each separate approach has some effects on muscle physiology, the combination of the dystrophin rescue and the downregulation of the myostatin receptor is required to massively improve both the tetanic force and the specific force. This study provides a novel pharmacogenetic strategy for treatment of certain neuromuscular diseases associated with muscle wasting.

Polarized mitochondria as guardians of NK cell fitness.

Distinct metabolic demands accompany lymphocyte differentiation into short-lived effector and long-lived memory cells. How bioenergetics processes are structured in innate natural killer (NK) cells remains unclear. We demonstrate that circulating human CD56Dim (NKDim) cells have fused mitochondria and enhanced metabolism compared with CD56Br (NKBr) cells. Upon activation, these 2 subsets showed a dichotomous response, with further mitochondrial potentiation in NKBr cells vs paradoxical mitochondrial fission and depolarization in NKDim cells. The latter effect impaired interferon-γ production, but rescue was possible by inhibiting mitochondrial fragmentation, implicating mitochondrial polarization as a central regulator of NK cell function. NKDim cells are heterogeneous, and mitochondrial polarization was associated with enhanced survival and function in mature NKDim cells, including memory-like human cytomegalovirus-dependent CD57+NKG2C+ subsets. In contrast, patients with genetic defects in mitochondrial fusion had a deficiency in adaptive NK cells, which had poor survival in culture. These results support mitochondrial polarization as a central regulator of mature NK cell fitness.

Ultrasound-Mediated Blood-Brain Barrier Opening Improves Whole Brain Gene Delivery in Mice.

Gene therapy represents a powerful therapeutic tool to treat diseased tissues and provide a durable and effective correction. The central nervous system (CNS) is the target of many gene therapy protocols, but its high complexity makes it one of the most difficult organs to reach, in part due to the blood-brain barrier that protects it from external threats. Focused ultrasound (FUS) coupled with microbubbles appears as a technological breakthrough to deliver therapeutic agents into the CNS. While most studies focus on a specific targeted area of the brain, the present work proposes to permeabilize the entire brain for gene therapy in several pathologies. Our results show that, after i.v. administration and FUS sonication in a raster scan manner, a self-complementary AAV9-CMV-GFP vector strongly and safely infected the whole brain of mice. An increase in vector DNA (19.8 times), GFP mRNA (16.4 times), and GFP protein levels (17.4 times) was measured in whole brain extracts of FUS-treated GFP injected mice compared to non-FUS GFP injected mice. In addition to this increase in GFP levels, on average, a 7.3-fold increase of infected cells in the cortex, hippocampus, and striatum was observed. No side effects were detected in the brain of treated mice. The combining of FUS and AAV-based gene delivery represents a significant improvement in the treatment of neurological genetic diseases.

Biocompatibility of four common orthopedic biomaterials following neuroelectromyostimulation: An in-vivo study.

Despite the worldwide high prevalence of total joint arthroplasty (TJA), life expectancy of prosthesis remains limited by mechanical and chemical constraint which promote wear debris production, surrounding tissues damage and finally prosthesis loosening. Such results could be amplified by neuro-myoelectrostimulation (NMES; widely used to reduce neuromuscular deficits observed following TJA surgery). It was previously described in an in vivo experiment that interactions between NMES and Ti6Al4V implant are deleterious for both implant and surrounding muscles. The purpose of the present study was to compare the biocompatibility of four common orthopedic biomaterials, two metallic (Ti6Al4V, CrCo) and two nonmetallic (PEEK, Al2 O3 ) alloys, fixed on rat tibial crest in which the surrounding muscles were electrostimulated. Muscle cell death rate was not found significantly increased, with or without electrical stimulation for nonmetallic implants. Contrary to Ti6Al4V alloy, the CrCo implant did not induce destruction of the surrounding muscle. However, cell viability decreased for both metallic alloys when NMES was applied but within a greater significant extent for Ti6Al4V implant. Otherwise, when NMES was applied, implant-to-bone adhesion significantly decreased for Ti6Al4V while no significant difference was found for PEEK, Al2 O3 , and CrCo. Statistical analyses reveal also a lesser adhesion strength for Ti6Al4V compared with CrCo when NMES was applied. Selecting the most suitable material in term of biocompatibility remains a major concern and non-metallic materials seems to be more appropriated in regard to electrical currents used for post TJA care. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1156-1164, 2018.

Titanium Implant Impairment and Surrounding Muscle Cell Death Following High-Salt Diet: An In Vivo Study.

AIM OF THE STUDY: High-salt consumption has been widely described as a risk factor for cardiovascular, renal and bone functions. In the present study, the extent to which high-salt diet could influence Ti6Al4V implant surface characteristic, its adhesion to rat tibial crest, and could modify muscle cell viability of two surrounding muscles, was investigated in vivo. These parameters have also been assessed following a NMES (neuro-myoelectrostimulation) program similar to that currently used in human care following arthroplasty. RESULTS: After a three-week diet, a harmful effect on titanium implant surface and muscle cell viability was noted. This is probably due to salt corrosive effect on metal and then release of toxic substance around biologic tissue. Moreover, if the use of NMES with high-salt diet induced muscles damages, the latter were higher when implant was added. Unexpectedly, higher implant-to-bone adhesion was found for implanted animals receiving salt supplementation. CONCLUSION: Our in vivo study highlights the potential dangerous effect of high-salt diet in arthroplasty based on titanium prosthesis. This effect appears to be more important when high-salt diet is combined with NMES.

Titanium implant impairment and surrounding muscle cell death following neuro-myoelectrostimulation: An in vivo study.

Electrical currents have deleterious effects on biomedical metallic implants. However, following arthroplasty, neuro-myoelectrostimulation (NMES) is often used in patient rehabilitation. Such a rehabilitation technique could compromise patient recovery through deleterious effects on metallic alloys and biological tissues. The purpose of our study was to assess the effects of NMES on a Ti6Al4V implant placed in a rat tibial crest and the surrounding muscle tissues. This in vivo study allowed to bring to the fore the prosthesis behavior under mechanical and electromagnetic loads induced by NEMS stimulation. After 3 weeks, implant-to-bone adhesion significantly decreased in stimulated animals compared with nonstimulated animals. Surface mapping indicated titanium implant degradation after NMES. Furthermore, NMES alone did not induce muscle damage contrary to that found in implanted animals. The muscle damage rate was significantly higher in implanted and stimulated animals compared with implanted-only animals. It seems obvious that rehabilitation programs using the NMES technique could induce early deterioration of biomaterial employed for surgical implants. Clinicians should reconsider the use of NMES as a rehabilitation technique for patients with titanium prostheses.

A new method to assess weight-bearing distribution after central nervous system lesions in rats.

The aim of the present study is to assess the relevance of weight-bearing distribution (DWB) measurement in freely moving rats after stroke and thoracic spinal cord injuries. Animals were divided in 2 experiments: (1) The middle cerebral artery occlusion-reperfusion (MCAO-r) experiment containing the MCAO group in which focal brain ischaemia was induced by transient MCA occlusion and (2) the thoracic hemisection experiment containing the TH group in which a spinal cord hemisection was performed at the T10 level. A Control and respective Sham groups were also included in each experiment. Not only the pressure exerted by each paw was measured but also different ratios such as: (1) the sum of the right and the left forepaws was normalized by the sum of the right and the left hindpaws (F/H), (2) the left forepaw was normalized by the right forepaw (LF/RF), (3) the left hindpaw was normalized by the right hindpaw (LH/RH). Additionally, the times spent on 3 paws and on 4 paws were measured. Only the time spent on 4 paws was shorter in the MCAO group than in the Control (p<0.001) and in the Sham (p<0.01) groups. The LH/RH ratio of the TH group at the 1st week was lower (p<0.01) than the pre-surgical value. Moreover, its F/H ratio was superior (p<0.001) to the ones of the Control and the Sham groups. Our study indicates that DWB should be more frequently used to evaluate both the severity of central nervous system traumas and the effectiveness of pharmacological and/or rehabilitation strategies.