Targeting caspase-6 and caspase-8 to promote neuronal survival following ischemic stroke.

Previous studies show that caspase-6 and caspase-8 are involved in neuronal apoptosis and regenerative failure after trauma of the adult central nervous system (CNS). In this study, we evaluated whether caspase-6 or -8 inhibitors can reduce cerebral or retinal injury after ischemia. Cerebral infarct volume, relative to appropriate controls, was significantly reduced in groups treated with caspase-6 or -8 inhibitors. Concomitantly, these treatments also reduced neurological deficits, reduced edema, increased cell proliferation, and increased neurofilament levels in the injured cerebrum. Caspase-6 and -8 inhibitors, or siRNAs, also increased retinal ganglion cell survival at 14 days after ischemic injury. Caspase-6 or -8 inhibition also decreased caspase-3, -6, and caspase-8 cleavage when assayed by western blot and reduced caspase-3 and -6 activities in colorimetric assays. We have shown that caspase-6 or caspase-8 inhibition decreases the neuropathological consequences of cerebral or retinal infarction, thereby emphasizing their importance in ischemic neuronal degeneration. As such, caspase-6 and -8 are potential targets for future therapies aimed at attenuating the devastating functional losses that result from retinal or cerebral stroke.

Uncoupling Neogenin association with lipid rafts promotes neuronal survival and functional recovery after stroke.

The dependence receptor Neogenin and its ligand, the repulsive guidance molecule a (RGMa), regulate apoptosis and axonal growth in the developing and the adult central nervous system (CNS). Here, we show that this pathway has also a critical role in neuronal death following stroke, and that providing RGMa to neurons blocks Neogenin-induced death. Interestingly, the Neogenin pro-death function following ischemic insult depends on Neogenin association with lipid rafts. Thus, a peptide that prevents Neogenin association with lipid rafts increased neuronal survival in several in vitro stroke models. In rats, a pro-survival effect was also observed in a model of ocular ischemia, as well as after middle cerebral artery occlusion (MCAO). Treatments that prevented Neogenin association with lipid rafts improved neuronal survival and the complexity of the neuronal network following occlusion of the middle artery. Toward the development of a treatment for stroke, we developed a human anti-RGMa antibody that also prevents Neogenin association with lipid rafts. We show that this antibody also protected CNS tissue from ischemic damage and that its application resulted in a significant functional improvement even when administrated 6 h after artery occlusion. Thus, our results draw attention to the role of Neogenin and lipid rafts as potential targets following stroke.

The repulsive guidance molecule, RGMa, promotes retinal ganglion cell survival in vitro and in vivo.

The repulsive guidance molecule, RGMa, and its receptor Neogenin, regulate neuronal cell death during development, but little is known about their expression and roles in the adult CNS. Here, we show that Neogenin is expressed in the adult rodent retina, particularly on retinal ganglion cells. To determine whether the Neogenin/RGMa pathway is important in the fully developed retina, we examined its contribution to damage-induced neurodegeneration. The effects of RGMa on survival of retinal ganglion cells (RGCs) were examined in vitro and in vivo. Using cultured whole-mount retinal explants, we showed that the addition of RGMa increased RGC survival and that this effect was mediated by the Neogenin receptor. Immunohistochemical analysis indicated that the inhibition of cell death by RGMa resulted from reduced caspase-3 activation. Then, using an in vivo model of RGC apoptosis after optic nerve transection, we demonstrated that intraocular injection of RGMa at 3 and 7 days after axotomy greatly reduced RGC death 14 days postaxotomy. This study provides the first evidence that RGMa is a molecular target for neuroprotection in retinal pathologies, and suggests that targeting "dependence receptors" such as Neogenin has therapeutic potential for the treatment of neuropathologies in the adult CNS.

Kv1.1 and Kv1.3 channels contribute to the degeneration of retinal ganglion cells after optic nerve transection in vivo.

Degeneration of retinal ganglion cells (RGCs) - an important cause of visual impairment - is often modeled by optic nerve transection, which leads to apoptotic death of these central nervous system neurons. With this model, we show that specific voltage-gated K(+) channels (Kv1 family) contribute to the degeneration of rat RGCs and expression of apoptosis-related molecules in vivo. Retinal expression of Kv1.1, Kv1.2, Kv1.3 and Kv1.5 was examined by quantitative real-time reverse transcriptase-PCR and immunohistochemistry. Kv channel blockers and channel-specific short-interfering RNAs (siRNAs) were used to assess their roles in RGC degeneration. We found that (i) rat RGCs express Kv1.1, Kv1.2 and Kv1.3 (but not Kv1.5); (ii) intraocular injection of agitoxin-2 or margatoxin, potent blockers of Kv1.1, Kv1.2 and Kv1.3 channels, dose-dependently reduced the RGC degeneration; (iii) siRNAs applied to the cut optic nerve were rapidly transported throughout RGCs only, in which they reduced the expression of the cognate channel only. Our results show differential roles of the channels; siRNAs directed against Kv1.1 or Kv1.3 channels greatly reduced RGC death, whereas Kv1.2-targeted siRNAs had only a small effect, and siRNAs against Kv1.5 were without effect. (iv) Kv1.1 and Kv1.3 channels apparently contribute to cell-autonomous death of RGCs through different components of the apoptotic machinery. Kv1.1 depletion increased the antiapoptotic gene, Bcl-X(L), whereas Kv1.3 depletion reduced the proapoptotic genes, caspase-3, caspase-9 and Bad.

Development and characterization of a hemorrhagic rat model of central post-stroke pain.

Stroke is the leading cause of disability in the industrialized world and it is estimated that up to 8% of stroke victims suffer from some form of central post-stroke pain (CPSP). Thalamic syndrome is form of central pain that typically results from stroke in the thalamus. In the present study, we describe the development and characterization of a rat model of thalamic CPSP. This model is based on a hemorrhagic stroke lesion in the ventral posterolateral nucleus of the thalamus, one of the reported causes of thalamic syndrome in humans. Behavioral analysis showed that animals displayed hyperesthesia in response to mechanical pinch stimulation, with sensitivity localized to the hind limb. This response appeared within 7 days of the intra-thalamic hemorrhage. Animals also showed increased thermal sensitivity in the contralateral hind limb. Histopathology indicated the presence of activated microglia adjacent to the core of hemorrhagic lesions in the thalamus. Neutrophils were confined to the hemorrhage core, indicating that they entered in the initial bleed. By 7 days, bands of activated microglia and astrocytes separated the hematoma from surviving neurons at the edge of the lesion. We did not observe any terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive neurons beyond the immediate hematoma at 1, 3, or 7 days after hemorrhage. Surviving neurons were located in the vicinity of activated microglia and astrocytes at the outer edge of the hematoma. Thus, thalamic hemorrhage produces a confined lesion that destroys the tissue within the initial bleed, with little or no neuron death beyond the hemorrhage core. Surviving neurons surrounded by activated glial cells likely contribute to neuropathic pain in this model. This thalamic hemorrhage model is useful for studying the neuropathology and physiology of thalamic syndrome, and developing therapeutics for central post-stroke pain.

[Ropivacaine infiltration during breast cancer surgery: postoperative acute and chronic pain effect]. / Infiltration de ropivacaïne en chirurgie carcinologique du sein: effet sur la douleur postopératoire aiguë et chronique.

OBJECTIVES: Decrease acute pain after breast cancer surgery by an infiltration of ropivacaine. Analyse effect on chronic pain. STUDY DESIGN: Prospective randomised double blind versus placebo study. PATIENTS AND METHODS: Eighty-one patients randomised between two groups received wound infiltration with 40 ml of ropivacaine 4.75 mg/ml or placebo. Acute pain was assessed during 24h with analogical visual scale and antalgic consumption. One year later, telephonic interviews looked for chronic pain and evaluate it with McGill Pain Questionnaire. RESULTS: Analogical visual scale pain score, antalgic consumption and chronic pain incidence were similar between groups. CONCLUSION: Ropivacaine scar infiltration provided no acute or chronic pain relief after breast cancer surgery.

The upregulation of GLAST-1 is an indirect antiapoptotic mechanism of GDNF and neurturin in the adult CNS.

Glial cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) protect retinal ganglion cells (RGCs) from axotomy-induced apoptosis. It is likely that neuroprotection by GDNF or NTN in the adult central nervous system (CNS) involves indirect mechanisms and independent signal transduction events. Extracellular glutamate is a trigger of apoptosis in injured RGCs, and glutamate transporter levels can be upregulated by GDNF. Therefore, GDNF may indirectly protect RGCs by enhancing glutamate uptake in the retina. We studied the upregulation of the glutamate transporters GLAST-1 and GLT-1 by GDNF and NTN, and the intracellular pathways required for GDNF/NTN neuroprotection. GDNF required phosphoinositide-3 kinase (PI3K) and Src activity to upregulate GLAST-1 and GLT-1. NTN required PI3K activity to upregulate GLAST-1 and did not affect GLT-1 levels. PI3K activity was also important for GDNF and NTN neuroprotection following optic nerve transection. However, GDNF also required Src and mitogen-activated protein kinase activity to prevent RGC apoptosis. RNA interference demonstrated that the upregulation of GLAST-1 by GDNF and NTN is required to rescue RGCs. Thus, additional independent signal transduction events, together with the upregulation of GLT-1 by GDNF, differentiate the biological activity of GDNF from NTN. Furthermore, the upregulation of the glial glutamate transporter GLAST-1 by both factors is an indirect neuroprotective mechanism in the CNS.

Effects of adenoviral-mediated gene transfer of interleukin-10, interleukin-4, and transforming growth factor-beta on the survival of axotomized retinal ganglion cells.

Nitric oxide, synthesized by reactive microglia and astrocytes has been implicated in promoting neuronal degeneration observed in many diseases and insults of the central nervous system. We have recently shown that inducible nitric oxide synthase is expressed by retinal glial cells following optic nerve transection and that inhibition of nitric oxide synthesis enhances the survival of injured retinal ganglion cells. Anti-inflammatory cytokines including interleukin-10 (IL-10), interleukin-4 (IL-4), and transforming growth factor-beta (TGF-beta) have been shown to prevent inducible nitric oxide synthase expression, and inhibit nitric oxide synthesis by microglia and astrocytes in culture. In the present study, we examined the effects of adenoviral mediated gene transfer of anti-inflammatory cytokines on the survival of axotomized retinal ganglion cells. Intraocular administration of adenoviral vectors encoding interleukin-10 (Ad.IL-10) and interleukin-4 (Ad.IL-4) enhanced the survival of axotomized retinal ganglion cells at 14 days after axotomy. Adenoviral vectors encoding TGF-beta (Ad.TGF-beta) had no effect on retinal ganglion cell survival. Separate animals were pretreated by injection of Ad.IL-10 or Ad.IL-4 into the superior colliculus (s.c.), the major target of ganglion cells, 7 days prior to axotomy. S.c. administration of Ad.IL-10 or Ad.IL-4 significantly increased ganglion cell survival compared with intraocular injection. IL-10 and IL-4 gene transfer also reduced the density of infiltrating ED1 positive monocytes in the nerve fiber layer at 14 days postaxotomy. Ad.TGF-beta increased the density of ED1 positive monocytes infiltrating the nerve fiber layer after axotomy. Vectors encoding IL-10 or IL-4 also decreased nitrotyrosine immunoreactivity in the inner retina at 7 days postaxotomy, suggesting that these cytokines protect retinal ganglion cells from peroxynitrite formation that results from nitric oxide synthesis by activated glial cells. The present study has implications for the treatment of CNS injury and diseases that involve reactive microglia and astrocytes. Our results suggest that interleukin-10 and interleukin-4 may help prevent neurodegeneration caused by the activation of glial cells after CNS injury.

[Severe obstetric complications nescessitating hospitalization and intensive care: a ten year retrospective study]. / Complications obstétricales graves nécessitant une hospitalisation en réanimation: étude rétrospective sur 10 ans au CHU de Besançon.

OBJECTIVES: To assess the serious maternal morbidity during pregnancy, delivery and post partum, which led to an hospitalization in a medical or surgical intensive care unit. STUDY DESIGN: A Retrospective study was carried out on a period of ten years, from July 1986 to July 1996, in the University Teaching Hospital of Besançon. PATIENTS: The criterions of inclusion come from the definition of the serious maternal morbidity decided by the Inserm: any admission of a pregnant woman in a medical or surgical intensive care unit in the 42 days of the post-partum, whatever the term of the pregnancy and the type of the post-partum, extra uterine pregnancy, spontaneous miscarriage and medical or voluntary abortion. METHODS: Forty-six patient's medical file hospitalized in a medical or surgical intensive care unit between July, 1st 1986 and July, 31st 1996, have been studied. RESULTS: The analysis of the cause underline the gravity of the pathologies handled with young patients and initially healthy, the short length of controlled ventilation and hospitalization, the avoidability of great number of transfer in an intensive care unit, and the lack of hospitalization due to anaesthesia. The frequency of hospitalisation in an intensive care unit during and after the pregnancy was estimated at 0.17% of lives births. CONCLUSION: The serious maternal morbidity could be an indicator of the quality of the obstetrics cares which would complete the study of the maternal mortality. The potential gravity of the complication of the pregnancy and the delivery require better care of this patients.

Increased TUNEL staining in brains of autoimmune Fas-deficient mice.

Profound changes in brain morphology and behavior coincide with the spontaneous development of systemic autoimmune/inflammatory disease in Fas-deficient MRL-lpr mice. The dendrites atrophy, the density of hippocampal and cortical neurons decreases, and an anxious/depressive-like behavior emerges while lymphoid cells infiltrate into the choroid plexus of MRL-lpr mice. We hypothesized that the inherited lack of the Fas-dependent anti-inflammatory mechanism would lead to unsuppressed immune activity, characterized by reduced apoptosis in the MRL-lpr brain. Using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeled (TUNEL) method as an indicator of apoptosis, a surprisingly high incidence of TUNEL-positive cells was observed in the hippocampus, choroid plexus and periventricular regions of MRL-lpr mice, 5-10-fold higher than that found in the MRL +/+ control brain. Immunostaining with anti-CD3, CD4 and CD8 monoclonal antibodies showed limited overlap between CD-positive and TUNEL-positive cells, suggesting that the dying cells are for the most part (approximately 70%) not T-lymphocytes. Although further characterization of the phenotype of the dying cells and the mechanism of cell death are required, the present results suggest the involvement of a Fas-independent apoptotic process in neurodegeneration induced by systemic autoimmune disease.

Nitric oxide synthase inhibition delays axonal degeneration and promotes the survival of axotomized retinal ganglion cells.

Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated in neuronal cytotoxicity following trauma to the central nervous system. The aim of the present study was to examine the role of NO in mediating axotomy-induced retinal ganglion cell (RGC) death. We observed increases in iNOS expression by microglia and Müller cells in the retina after optic nerve transection. This was paralleled by the induced expression of constitutive NOS (cNOS) in RGCs which do not normally express this enzyme. In order to determine if NO is cytotoxic to axotomized RGCs, the nonspecific NOS inhibitors Nomega-nitro-L-arginine (NOLA) or N-nitro-L-arginine methyl ester (L-NAME) were delivered to the vitreous chamber by intraocular injections. Both NOLA and L-NAME significantly enhanced RGC survival at 7, 10, and 14 days postaxotomy. The separate contributions of iNOS and cNOS to RGC degeneration were examined with intraocular injections of the specific iNOS inhibitor L-N(6)-(I-iminoethyl)lysine hydrochloride or the specific cNOS inhibitor L-thiocitrulline. Our results suggest that cNOS plays a greater role in RGC degeneration than iNOS. In addition to enhancing RGC survival, NOS inhibitors delayed the retrograde degeneration of RGC axons after axotomy. We conclude that NO synthesized by retinal iNOS and cNOS plays a major role in RGC death and retrograde axonal degeneration following axotomy.

Effects of GDNF on retinal ganglion cell survival following axotomy.

Recent evidence suggests that approximately 90% of retinal ganglion cells (RGCs) die by the process of apoptosis within 14 days of optic nerve transection. RGCs begin to disappear from the retina between 5 and 7 days postaxotomy when the highest percentage of RGCs show characteristics typical of apoptosis. A single intraocular injection of glial cell-line derived neurotrophic factor (GDNF) given at the time of axotomy resulted in a delay in the initiation of RGC death and increased the densities of surviving RGCs at 7, 10 and 14 days postaxotomy. The mean RGC densities in GDNF treated retinas at 7 (2381 +/- 144), 10 (1561 +/- 117) and 14 (1123 +/- 116) days postaxotomy were significantly higher than that of controls (1835 +/- 82, 835 +/- 272 and 485 +/- 39, respectively). The loss of RGCs was paralleled by increases in TUNEL positive staining in control retinas and a lower percentage of TUNEL positive cells in GDNF treated retinas at 5, 7 and 10 days postaxotomy. These results suggest that GDNF is capable of promoting RGC survival following injury, possibly by interfering with an essential step in apoptosis.

[A rare cause of postpartum hemorrhage: a genital thrombus]. / Une cause rare d'hémorragie du post-partum: le thrombus génital.

Puerperal hematoma is a grave but fortunately rare hemorrhagic post-partum complication (occurring in less than 1:1000 deliveries). The hematoma arises due to detachment of para-vaginal conjunctive tissue. In this type of tissue, no natural hemostasis take place and the hematoma may spread into the retroperitoneal cavity. The different risk factors include primiparity, instrumental extraction of the foetus, pre-eclampsia, twin pregnancy and the presence of vulvo-vaginal varicose veins. External bleeding may not always be evident and other clinical symptoms may be delayed. Despite this, rapid course may still occur with drastic consequences. When a case is referred, an examination of the vulvo-vaginal region is mandatory, resuscitation and surgery performed immediately. If this fails angiographic embolization should be carried out. The prognostic outcome of this rare case of post-partum hemorrhagia is highly dependent on early diagnosis and rapid treatment involving close cooperation between obstetricians and anesthetists, and also of rapid embolization to prevent possible intractable hematomas.

[Hereditary angioneurotic edema in gynecology-obstetrics. Management]. / L'oedème angioneurotique héréditaire en gynécologie-obstétrique. Conduite à tenir.

Hereditary angioneurotic edema is a rare disease (250 cases currently identified in France). It is the most frequent among hereditary deficiencies of the complement system. This severe disease is fatal in 15-20% of the patients before 40 years of age. Death can be prevented if correct steps are taken in case of even minor trauma, which often trigger acute, potentially fatal, episodes. Danazol is used as preventive therapy before surgery and C1-esterase inhibitor or fresh frozen plasma is given for acute episodes. In case of obstetrical trauma, C1-esterase inhibitors should be used either as prophylaxy or as treatment. Fresh frozen plasma is used if C1-esterase inhibitors are not available. We report here our experience with three patients with known hereditary angioneurotic edema. Gynecology surgery was required in two.

[Obstetrical epidural analgesia: an epidemiological in Bourgogne and Franche-Comté 1993]. / Analgésie péridurale obstétricale: étude épidémiologique en 1993 en Bourgogne et en Franche-Comté.

Many publications report the number of epidural analgesias carried out for the last years in English-speaking countries. On the other hand, very little information exists about the incidence of obstetrical epidural analgesia performed in South and Central Europe, particularly in France. A retrospective study within the regions of Bourgogne and Franche-Comté was carried out for the year 1993 firstly, to determine the number of obstetrical epidural analgesias performed and the drugs used; and secondly, to describe the organization and the problems encountered. The result of this study leads to one major question: what is the best type of organization in order to guarantee the highest safety, given the increasing demand for epidural analgesia in obstetrics?

[Hemodynamic effect of intrathecal clonidine]. / Retentissement hémodynamique de la clonidine intrathécale.

Intrathecal administration of bupivacaine and clonidine results in a significant prolongation of both motor and sensitive blocks but side effects-hypotension and bradycardia-are observed. We compared two groups of patients allocated randomly. One group received intrathecal bupivacaine-morphine and the other one received clonidine-bupivacaine-morphine. In the group which received clonidine a blood pressure decrease was observed from 2nd to 8th hour, independently from the sympathetic block and easily corrected by i.v. ephedrine. Some possible explanations are discussed. No complications due to clonidine were observed.

[Angioneurotic edema and anesthesia: preparation and perioperative monitoring]. / Oedème angioneurotique et anesthésie: préparation et surveillance périopératoire.

Hereditary angioneurotic oedema is an autosomal dominant disease associated with serum deficiency of functional C1-inhibitor. It is characterized by periodic swelling of subcutaneous tissues, abdominal viscera and upper airways. Lethal acute episodes of oedema can occur during anaesthesia and surgery. It is essential to prepare such patients before surgery. This article describes three cases (kidney transplantation, caesarean section, normal delivery) and the various preventive measures used to avoid acute episodes during anaesthesia and surgery. Antibrinolytic agents, androgens, fresh frozen plasma, C1-inhibitor concentrate can be administered. Their various indications are discussed.

[Postoperative analgesia by intrathecal morphine and respiratory depression: value of low doses (apropos of 285 cases)]. / Analgésie postopératoire par morphine intrathécale et dépression respiratoire: intérêt des faibles doses (à propos de 285 cas).

Morphine at very low dose gives a good post-operatory analgesia without major secondary effects. This study analyses retrospectively 285 spinal anaesthesia with hyperbaric 0.5% bupivacaine 0.2 mg.kg-1 and morphine 0.25 mg in adult urologic surgery. The analysis of enquiry in analgesic during the post-operatory period shows that in most cases (72%) the patients have supported the first 24 hours without any complementary analgesia; 28% of patients needs a complementary analgesia realized with 1 g of paracetamol. Intravenous morphinics have never been necessary in post-operative period. The analgesia was adequate for the patient's comfort, and it never mask a surgery complication. No respiratory complication appeared, even in 13 patients who needed intravenous morphinics in per-operative.

[Spinal anesthesia at T12 or T10 level with hyperbaric bupivacaine 0.5%: value of determining the useful dosage according to the weight]. / Rachianesthésie de niveau T12 ou T10 avec la bupivacaïne à 0.5% hyperbare: intérêt de fixer la dose utile en fonction du poids.

A retrospective study was carried out on anaesthetic records concerning spinal anaesthesia with hyperbaric bupivacaine 0.5% in urologic surgery. Three doses were utilised: slight (< 0.19 mg.kg-1), mean (0.19-0.21 mg.kg-1) or important (> 0.21 mg.kg-1) for two different levels: T12 or T10. Important doses may be involved excessive extension. Failures are perhaps more frequent with slight doses. Mean doses, 0.20 mg.kg1, seems to be recommended.