Targeting the splicing of mRNA in autoimmune diseases: BAFF inhibition in Sjögren's syndrome as a proof of concept.

BAFF (B-cell-activating factor of the tumor necrosis factor family), a pivotal cytokine for B-cell activation, is overexpressed by salivary gland (SG) epithelial cells in primary Sjogren's syndrome (pSS). ΔBAFF, a physiological inhibitor of BAFF, is a minor alternative splice variant of BAFF. A U7 RNA was reengineered to deliver antisense sequences targeting BAFF splice regions. A major decrease of BAFF messenger RNA (mRNA) and protein secretion, concomitantly with the increase of ΔBAFF mRNA, was observed in vitro. In vivo, SG retrograd instillation of nonobese diabetic mice by the modified U7 cloned into an adeno-associated virus vector significantly decreased BAFF protein expression and lymphocytic infiltrates and improved salivary flow. This study offers a rationale for localized therapeutic BAFF inhibition in pSS and represents a proof of concept of the interest of exon skipping in autoimmune diseases.

Muco-ciliary differentiation of nasal epithelial cells is decreased after wound healing in vitro.

BACKGROUND: Epithelial damage and modifications of cell differentiation are frequent in airway diseases with chronic inflammation, in which transforming growth factor-beta1 (TGF-beta1) plays an important role. The aim of this study was to evaluate the differentiation of human nasal epithelial cells (HNEC) after wound healing and the potential effects of TGF-beta1. METHODS: Basal, mucus, and ciliated cells were characterized by cytokeratin-14, MUC5AC, and betaIV tubulin immunodetection, respectively. Their expression was evaluated in situ in nasal polyps and in an in vitro model of wound healing in primary cultures of HNEC after wound closure, under basal conditions and after TGF-beta1 supplementation. Using RT-PCR, the effects of TGF-beta1 on MUC5AC and DNAI1 genes, specifically transcribed in mucus and ciliated cells, were evaluated. RESULTS: In situ, high TGF-beta1 expression was associated with low MUC5AC and betaIV tubulin expression. In vitro, under basal conditions, MUC5AC expression remained stable, cytokeratin-14 expression was strong and decreased with time, while betaIV tubulin expression increased. Transforming growth factor-beta1 supplementation downregulated MUC5AC and betaIV tubulin expression as well as MUC5AC and DNAI1 transcripts. CONCLUSION: After a wound, differentiation into mucus and ciliated cells was possible and partially inhibited in vitro by TGF-beta1, a cytokine that may be involved in epithelial remodeling observed in chronic airway diseases.

Expression of vesicular glutamate transporters, VGLUT1 and VGLUT2, in cholinergic spinal motoneurons.

Mammalian spinal motoneurons are cholinergic neurons that have long been suspected to use also glutamate as a neurotransmitter. We report that VGLUT1 and VGLUT2, two subtypes of vesicular glutamate transporters, are expressed in rat spinal motoneurons. Both proteins are present in somato-dendritic compartments as well as in axon terminals in primary cultures of immunopurified motoneurons and sections of spinal cord from adult rat. However, VGLUT1 and VGLUT2 are not found at neuromuscular junctions of skeletal muscles. After intracellular injection of biocytin in motoneurons, VGLUT2 is observed in anterogradely labelled terminals contacting Renshaw inhibitory interneurons. These VGLUT2- and VGLUT1-positive terminals do not express VAChT, the vesicular acetylcholine transporter. Overall, our study establishes for the first time that (i) mammalian spinal motoneurons express vesicular glutamate transporters, (ii) these motoneurons have the potential to release glutamate (in addition to acetylcholine) at terminals contacting Renshaw cells, and finally (iii) the VGLUTs are not present at neuromuscular synapses of skeletal muscles.

Muscle precursor cell autografting in a murine model of urethral sphincter injury.

OBJECTIVE: To determine whether muscle precursor cells (MPCs) harvested from limb skeletal muscle can enhance the regeneration process of the striated urethral sphincter after injury. MATERIAL AND METHODS: Striated urethral sphincters of male mice were injured by an injection of a myotoxic substance (notexin). In the experimental group, 2 days after injury, MPCs were enzymatically harvested from striated muscles of the lower limbs and labelled with PKH 26, then immediately re-injected into the injured urethral sphincter of the same animal. In the control group, saline buffer was injected instead of MPCs. Animals were killed 7 days or 1 month after injury and the sphincters removed for histological study (the presence of PKH 26-labelled myofibres, measurement of myofibre diameter and total number of myofibres). RESULTS: MPC autografting accelerated sphincter muscle repair, as shown by a higher myofibre diameter (P = 0.03) and number (P = 0.01) in the experimental group than in the controls at 7 days. One month after their injection MPCs were still detectable in the regenerating sphincters and participated in the formation of new myofibres. CONCLUSION: This study provides the experimental basis for a new therapeutic approach to urethral sphincter insufficiency after surgical or obstetrical injury, based on MPC autografting.

Oligoclonal T-cells in blood and target tissues of patients with anti-Hu syndrome.

T-cell clones of unknown significance (TCUS), assessed by monoclonal or oligoclonal T-cell patterns in PCR-DGGE, were detected in blood of 7/9 patients with anti-Hu syndrome. Clonal patterns were also detected in 2/2 neoplastic lymph nodes, and in 2/2 inflamed dorsal root ganglia from three patients. Only some T-cell clones found in target tissues were also detected in blood or non-target tissues, and likely corresponded to TCUS. In one patient, an identical T-cell clone was found in both neoplastic lymph node tissue and dorsal root ganglia, but not in blood. Dorsal root-infiltrating lymphocytes were cytotoxic CD8(+) TIA-1(+) T-cells. They were often found in close contact to sensory neurons, most of which expressed MHC-1. Taken together, these data support a direct effector role of cytotoxic CD8(+) T-cells, the same clones being likely operative in sensory neuron damage and immune-mediated tumor growth control.

Macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle.

Macrophagic myofasciitis (MMF) is an emerging condition of unknown cause, detected in patients with diffuse arthromyalgias and fatigue, and characterized by muscle infiltration by granular periodic acid-Schiff's reagent-positive macrophages and lymphocytes. Intracytoplasmic inclusions have been observed in macrophages of some patients. To assess their significance, electron microscopy was performed in 40 consecutive cases and chemical analysis was done by microanalysis and atomic absorption spectrometry. Inclusions were constantly detected and corresponded to aluminium hydroxide, an immunostimulatory compound frequently used as a vaccine adjuvant. A lymphocytic component was constantly observed in MMF lesions. Serological tests were compatible with exposure to aluminium hydroxide-containing vaccines. History analysis revealed that 50 out of 50 patients had received vaccines against hepatitis B virus (86%), hepatitis A virus (19%) or tetanus toxoid (58%), 3-96 months (median 36 months) before biopsy. Diffuse myalgias were more frequent in patients with than without an MMF lesion at deltoid muscle biopsy (P < 0.0001). Myalgia onset was subsequent to the vaccination (median 11 months) in 94% of patients. MMF lesion was experimentally reproduced in rats. We conclude that the MMF lesion is secondary to intramuscular injection of aluminium hydroxide-containing vaccines, shows both long-term persistence of aluminium hydroxide and an ongoing local immune reaction, and is detected in patients with systemic symptoms which appeared subsequently to vaccination.

Differential microglial response to progressive neurodegeneration in the murine mutant Wobbler.

Activated microglia is associated with neurodegenerative processes, but the precise role of this cell population is difficult to identify. Most experimental models employed to examine microglial responses involve acute alterations of neuronal integrity, in contrast to the progressive nature of neurodegenerative diseases. In order to approach the clinical situation better, the microglial response was analyzed in the murine mutant Wobbler, which exhibits a well-characterized neurodegenerative pathology, manifested by motoneuronal death following a period of cellular dysfunction with characteristic morphological features. Microglial cells were identified using anti-Mac1 or anti-IgG antibodies. Examination of the changes in density, localization, and phenotype of microglia differentiated two types of responses in Wobblers. A first type of response was observed as early as in the third week after birth, when the only apparent neuronal defect was the morphological alteration of a subset of motoneurons in the cervical spinal cord, which was maintained later on. The activated microglia extended long processes that selectively ensheathed vacuolated motoneurons. At later stages, when motoneuron death became prominent, an additional type of response was characterized by an increased density of reactive microglia that was seen extending throughout the cervical enlargement. This secondary microglial response occurred in parallel to the infiltration of T-lymphocytes. Thus, these results point to a differential response of microglial cells to a progressive neurodegenerative process.

Ciliary neurotrophic factor may activate mature astrocytes via binding with the leukemia inhibitory factor receptor.

Ciliary neurotrophic factor (CNTF) acts on immature astrocytes that express its trimeric receptor. In contrast, mature astrocytes do not significantly express the specific CNTFalpha receptor subunit, yet they respond to CNTF administration in vivo. Here we show that this controversy may be solved by a shift in astroglial sensitivity to CNTF over time, related to a change in the type of receptor bound by the cytokine on mature astrocytes. A convergent set of results supports the hypothesis that the CNTF effect is due to the illegitimate binding on the leukemia inhibitory factor receptor (LIFR): (i) it requires high concentration of recombinant rat CNTF; (ii) it involves the Jak/Stat and Ras-MAPK pathways; (iii) it is preserved in CNTFRalpha-/- cells; (iv) it is potentiated by soluble CNTFRalpha added to the medium; and (v) it is significantly decreased by a partial antagonist of LIFR. On these bases, we propose a mechanistic model in which, in the adult brain, a CNTF/LIFR interglial system may be modulated by neurons that synthesize CNTFRalpha.

Angiotensin II induces nuclear factor- kappa B activation in cultured neonatal rat cardiomyocytes through protein kinase C signaling pathway.

Rat neonatal ventricular cardiomyocytes (RNVM) possess G protein-coupled AT(1)receptors for angiotensin II (AngII) that activate multiple intracellular pathways. To elucidate potential signaling mechanisms involved, we focussed on the nuclear transcription factor-kappa B (NF- kappa B) in RNVM culture. Using specific antibody to NF- kappa Bp65, immunolocalization of NF- kappa B was cytoplasmic in unstimulated cardiomyocytes, whereas NF- kappa B was translocated into the RNVM nucleus in response to AngII. This translocation was inhibited in the presence of calphostin C, a specific inhibitor of protein kinase C (PKC). Western blot analysis showed an increase of NF- kappa B in AngII-stimulated cardiomyocyte nuclear extracts as compared to controls. Biomolecular interaction analysis (BIA analysis) of NF- kappa B activation showed that only AngII-nuclear extracts bound to NF- kappa B consensus sequence with a high degree of affinity. This DNA-binding capacity was completely lost in calphostin C-treated cells. At transcriptional level in RNVM, AngII mediates the upregulation of matrix gelatinase (MMP-9), which is totally inhibited by calphostin C treatment. In conclusion, cardiomyocyte nuclear NF- kappa B translocation in response to Ang II via PKC pathway activates cardiomyocyte-specific transcription of MMP-9 and may activate transcription from responsive genes which are involved in cardiac hypertrophy process and/or cardiac remodeling.

Peripheral injections of Freund's adjuvant in mice provoke leakage of serum proteins through the blood-brain barrier without inducing reactive gliosis.

Breakdown of the blood-brain barrier (BBB) and ensuing gliosis are common events following physical trauma to the central nervous system (CNS) or during autoimmune diseases such as experimental allergic encephalomyelitis (EAE). Some studies of EAE in rodents report that peripheral injections of complete Freund's adjuvant (CFA), which contains heat-inactivated Mycobacterium to provoke peripheral inflammation without adversely affecting the CNS, can itself lead to increased BBB permeability to small tracer molecules and certain serum proteins. To study the equivocal relationship between serum protein extravasation and reactive gliosis, we injected C57BL/6 mice with CFA and histologically assessed the permeability of various serum proteins and the reactivity of proximal microglia and astrocytes in the uninjured brainstem and spinal cord enlargements after 1-4 weeks. Our results confirm that CFA injections induce progressive increases in the perivascular extravasation of serum IgG, albumin, IgM, and exogenous horseradish peroxidase, all to varying degrees, most prominently in the brainstem and cervical spinal cord after 2-3 weeks. More importantly, neither microglial cells nor astrocytes in regions of focal serum protein leakage appeared morphologically reactive based on immunoreactivity for CR3 receptors (Mac-1) or glial fibrillary acidic protein (GFAP), respectively. Because we found no evidence of T cell infiltration accompanying the exudates, our results indicate that in the absence of physical trauma or inflammatory cells resident CNS neuroglia remain quiescent upon exposure to extravasated serum proteins.

Altered balance between matrix gelatinases (MMP-2 and MMP-9) and their tissue inhibitors in human dilated cardiomyopathy: potential role of MMP-9 in myosin-heavy chain degradation.

BACKGROUND: End-stage of human dilated cardiomyopathy (DCM) is characterized by myocyte loss and fibrosis, and associated with ventricular dilatation and reduced cardiac function. Matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs) have been involved in the myocardial remodeling. AIMS: To evaluate the potential role of matrix gelatinases (MMP-2 and MMP-9) in DCM, the balance between gelatinases and TIMPs and the gelatinase localization were investigated in left free wall ventricles from six normal donors and six patients with DCM at the transplantation time. METHODS: TIMP-(1, 2, 3 and 4) mRNAs were analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). TIMP-1 and -2 protein content was assessed by ELISA. MMP-2 and MMP-9 expression were examined by zymography and immunological techniques. RESULTS: All TIMPs were down-regulated in DCM hearts, especially TIMP-1 (reduced by 80%). Gel zymography revealed similar activity of MMP-2 and MMP-9 in both tissues. By in situ zymography and immunohistochemistry, active and immunoreactive gelatinases were pericardiomyocyte in control hearts and intracardiomyocyte in DCM hearts. Intracellular MMPs were associated with sarcomeric structure in DCM. To estimate a putative role of these gelatinases, several sarcomeric contractile proteins were digested in vitro by purified active MMP-9. Only myosin-heavy chain was cleaved in vitro giving 180-, 120-, 80- and 20-kDa proteolytic fragments. In vivo, two major myosin-heavy chain proteolytic fragments (80 and 20 kDa) were detected by specific monoclonal antibody against myosin-heavy chain in DCM left ventricular homogenates, only. CONCLUSIONS: Taken together, these data highly suggest that MMP-2 and MMP-9 may be involved in the disorganization of the contractile apparatus in DCM hearts.

Heart and lung VEGF mRNA expression in rats with monocrotaline- or hypoxia-induced pulmonary hypertension.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is upregulated during exposure to hypoxia. In this study, we analyzed heart and lung VEGF mRNA expression and examined pulmonary vascular remodeling as well as myocardial capillary density in two rat models of pulmonary hypertension involving exposure to chronic hypoxia (CH) and treatment with monocrotaline (MCT), respectively. The rats were studied after 0.5, 1, 3, 15, and 30 days of exposure to 10% O2 or 1, 6, and 30 days after a subcutaneous MCT injection (60 mg/kg). Both CH and MCT induced pulmonary hypertension and hypertrophy of the right ventricle (RV) with increased RV weight and atrial natriuretic peptide mRNA expression. VEGF mRNA expression as assessed by Northern blot analysis was potently induced after 12 h of hypoxia in both the right and left ventricles. After prolonged exposure to hypoxia, VEGF mRNA returned to baseline in the left ventricle (LV) but remained increased in the RV, where it peaked after 30 days. In MCT rats, VEGF mRNA was unchanged in the LV but decreased by 50% in the RV and by 90% in the lungs after 30 days. VEGF mRNA remained unchanged in the lungs from CH rats. Pulmonary vascular remodeling was more pronounced in MCT than in CH rats. The number of capillaries per RV myocyte was increased in rats exposed to 30 days of hypoxia, whereas it remained unchanged in MCT rats despite a similar degree of RV hypertrophy. Our results suggest that the sustained increase in VEGF expression in the hypertrophied RV during CH may account for the increased number of capillaries per myocyte. In contrast, reduced VEGF expression in the lungs and RV of MCT rats may aggravate pulmonary vascular remodeling and compromise RV myocardial perfusion.

Regulation of growth factor gene expression in degenerating motoneurons of the murine mutant wobbler: a cellular patch-sampling/RT-PCR study.

Motoneuronal degenerative diseases are characterized by their progressivity; once affected, the motoneurons remain in altered states during an intermediate phase of degeneration prior to their final disappearance. Whether this survival period coincides with active metabolic rearrangements in the affected neuron remains unknown. As a first step toward the elucidation of this question, we developed cDNA pooled samples obtained from degenerating and control motoneuron mRNA populations through cellular patch sampling and RT-PCR, using the murine wobbler mutant as a model of spinal atrophy. Hybridization of the cDNA pools to various markers of intact or degenerating motoneurons allowed us to verify the cellular specificity of the patch sampling and indicated conservation of the original mRNA population complexity. Exploration of transcriptional alterations of genes encoding growth factors thought to be involved in motoneuronal development revealed that gene expression of the neurotrophin BDNF was induced in affected motoneurons, while expression of neurotrophin-3 was present in both neuronal types. Likewise, expression of a member of the epidermal growth factor (EGF) family, the neuregulin transcript sensory motor neuron-derived factor, was detected in both control and degenerating motoneurons, while transforming growth factor alpha, the functional homolog of EGF, was present only in the affected motoneurons. Immunohistochemical detection of corresponding proteins corroborated these observations. These results demonstrate that, during the course of their degeneration, motoneurons can initiate expression of novel genes which lead to the production of molecules endowed with trophic and/or differentiative properties for the neurons themselves and their glial environment. They also validate the use of the developed cDNA pooled samples for further exploration of transcriptional alterations taking place in degenerating motoneurons.

Abducens palsy after an intrathecal glucocorticoid injection. Evidence for a role of intracranial hypotension.

We report a case of abducens palsy eight days after an intrathecal glucocorticoid injection followed by post-lumbar puncture syndrome. T1-weighted magnetic resonance imaging scans showed marked diffuse postgadolinium enhancement of the supra- and infratentorial meninges consistent with intracranial hypotension syndrome. The palsy resolved almost completely and a repeat magnetic resonance imaging scan done after four months was normal. The mechanism of the meningeal thickening and contrast enhancement is discussed.

Phenotypic alteration of astrocytes induced by ciliary neurotrophic factor in the intact adult brain, As revealed by adenovirus-mediated gene transfer.

Synthesis of the ciliary neurotrophic factor (CNTF) and its specific receptor (CNTFRalpha) is widespread in the intact CNS, but potential biological roles for this system remain elusive. Contradictory results have been obtained concerning a possible effect on the morphological and biochemical phenotype of astrocytes. To reassess this question, we have taken advantage of adenovirus-mediated gene transfer into the rat brain to obtain the local release of CNTF. Stereotaxic administration of CNTF recombinant adenovirus vectors into the striatum led to phenotypic changes in astrocytes located in regions that were related axonally to striatal neurons at the injection site. Astrocytes appeared hypertrophied and displayed an increase in both GFAP and CNTF immunoreactivity. This response was observed up to 5 weeks after injection, the longest time studied. It was not observed after the administration of a control vector. The methodology used in the present study, allowing us to analyze the effect of the factor in areas remote from the injection site, has provided conclusive evidence that CNTF affects the astroglial phenotype in the intact CNS. The characteristics of these effects may explain why contradictory results have been obtained previously, because this signaling system seems to have a low efficiency and therefore requires a high local concentration of the factor close to the target cells. One might speculate as to the involvement of a CNTF astroglio-astroglial signaling system in the organized response of a population of astrocytes to changes in CNS homeostasis detected locally, even by a single cell.

Transforming growth factor alpha expression as a response of murine motor neurons to axonal injury and mutation-induced degeneration.

We previously showed that degenerating adult motor neurons of the murine mutant wobbler, a model of spinal muscular atrophy, express Transforming Growth Factor alpha (TGF alpha), a growth factor endowed with glio- and neurotrophic activities. Here, we evaluated whether TGF alpha expression is a general response of adult motor neurons to injury. Synthesis of its precursor (pro-TGF alpha) was investigated in another model of motoneuronal degeneration, the murine mutant muscle deficient, and in hypoglossal motor neurons following axonal crush and cut. In control conditions, motor neurons were devoid of pro-TGF alpha immunoreactivity. In the mutant lumbar spinal cord, pro-TGF alpha immunoreactive motor neurons appeared as soon as the disease developed and pro-TGF alpha expression persisted until the latest stages of degeneration. Motor neurons and astrocytes of the white matter weakly immunoreactive for the TGF alpha receptor were also present in both control and mutant lumbar spinal cords. Following hypoglossal nerve crush and cut, motoneuronal pro-TGF alpha expression was precocious and transient, visible at one day post-injury and lasting for only 3 days, during which time astrocyte-like cells immunoreactive for both TGF alpha and its receptor appeared within the injured nucleus. Enhanced TGF alpha mRNA levels following nerve crush showed that activation occurred at the transcriptional level. These results show that upregulation of TGF alpha is an early and common response of adult murine motor neurons to injury, regardless of its experimental or genetic origin.

Bcl-2 sensitivity differentiates two pathways for motoneuronal death in the wobbler mutant mouse.

The molecular events leading to motoneuronal death are still poorly understood. In mammals, the bcl-2 proto-oncogene, which encodes a membrane-associated protein, has been shown to suppress both developmental motoneuronal death and experimental axotomy-induced motoneuronal death. We assessed a potential protective effect of Bcl-2 on pathological motoneuronal death processes in adult rodents. We took advantage of the murine mutant wobbler, which undergoes progressive degeneration of the spinal and brainstem motoneurons. A hybrid carrying both the wobbler mutation and the human bcl-2 transgene under the control of the neuron-specific enolase promoter was produced. Although Bcl-2 protected spinal and brainstem motoneurons from developmental death and the postnatal motoneurons of the facial nucleus from axotomy-induced death, the pathological motoneuronal death was not altered in the adult hybrid. These results demonstrate that Bcl-2 sensitivity distinguishes at least two different motoneuronal death pathways in the wobbler mutant. They support the hypothesis that experimental and pathological motoneuronal death are dependent on different cellular mechanisms.

Short increase of BDNF messenger RNA triggers kainic acid-induced neuronal hypertrophy in adult mice.

Neurotrophin gene expression in adult brain varies according to physiological activity and following brain injury, suggesting a role in neuronal maintenance and plasticity. However, the exact roles and mechanisms of action of neurotrophins in the adult brain are still poorly understood. We have recently demonstrated that neurons of the adult mouse dentate gyrus can develop a conspicuous morphogenetic response to intrahippocampal injection of kainic acid. This response is correlated with long-lasting overexpression of the brain-derived neurotrophic factor gene, suggesting a causal relationship between molecular and structural changes. To test this hypothesis, brain-derived neurotrophic factor messenger RNA were sequestered in vivo by administration of antisense oligodeoxynucleotides. When administered before 20 h post-kainate, antisense oligodeoxynucleotides totally prevented the kainate-induced neuronal hypertrophy, while sense or missense sequences had no effect. On the other hand, the hypertrophic response was observed when antisense administration was begun 24 h post-kainate, indicating an involvement of brain-derived neurotrophic factor messenger RNA in the initiation of structural changes, but not in their evolution. The hypertrophy was blocked by inhibition of tyrosine kinase activities by K252a, suggesting an involvement of Trk high affinity receptors. Administration of human recombinant brain-derived neurotrophic factor without previous treatment by kainate failed to induce any morphogenetic response. These results show that a short activation of the brain-derived neurotrophic factor gene can, in association with neuronal activation by kainate, trigger dramatic and long-lasting morphological changes in adult neurons. A physiological role of brain-derived neurotrophic factor in adult brain could therefore be to link, by autocrine/paracrine action, activation of glutamate receptors and neuronal morphological adaptive responses.

Magnetic resonance angiographic analysis of atlanto-axial rotation: anatomic bases of compression of the vertebral arteries.

The aim of this study was to identify the functional anatomic factors involved in the maintenance or disturbance of flow in the vertebral aa. during atlanto-axial rotation. Fourteen healthy volunteers were studied by magnetic resonance angiography (MRA) by a three-dimensional sequence in phase contrast centered on the vertebral aa. at the level of the cranio-cervical junction before and after left rotation of the head. A decrease in the signal intensity of the arterial flow was sought for. The results were compared to the posterolateral development of the loop of the vertebral a. in its atlanto-axial segment in neutral position, and to the measurement of the angular opening between the atlas and axis in dynamic position. Seven subjects also had a three-dimensional CT study (3D CT) of the bony relations of C1 and C2 after rotation. In 4 subjects a disturbance of flow in the right vertebral a. was observed in the transverse foramen of C2. This occurred when two factors were combined: an under-developed atlanto-axial arterial loop and a C1-C2 angle exceeding 35 degrees in maximal rotation. In the other subjects a well-developed arterial loop and/or a C1-C2 angle of less than 35 degrees in maximal rotation were factors preserving the arterial flow. The risk factor associated with the C1-C2 angle seemed correlated in 3D CT with loss of the usual asymmetric character of rotation. A clinical application is reported with a case combining chronic rotational dysfunction of the cranio-cervical junction as shown by 3D CT and complete compression of the vertebral a. in MRA, confirmed by conventional angiography. A knowledge of this physiopathologic mechanism allows clinical detection and evaluation of the risk of any effect of pathology of the cranio-cervical junction on the vertebral a.