Characterization of nerve and microvessel damage and recovery in type 1 diabetic mice after permanent femoral artery ligation.

Neuropathy is the most common complication of the peripheral nervous system during the progression of diabetes. The pathophysiology is unclear but may involve microangiopathy, reduced endoneurial blood flow, and tissue ischemia. We used a mouse model of type 1 diabetes to study parallel alterations of nerves and microvessels following tissue ischemia. We designed an easily reproducible model of ischemic neuropathy induced by irreversible ligation of the femoral artery. We studied the evolution of behavioral function, epineurial and endoneurial vessel impairment, and large nerve myelinated fiber as well as small cutaneous unmyelinated fiber impairment for 1 month following the onset of ischemia. We observed a more severe hindlimb dysfunction and delayed recovery in diabetic animals. This was associated with reduced density of large arteries in the hindlimb and reduced sciatic nerve epineurial blood flow. A reduction in sciatic nerve endoneurial capillary density was also observed, associated with a reduction in small unmyelinated epidermal fiber number and large myelinated sciatic nerve fiber dysfunction. Moreover, vascular recovery was delayed, and nerve dysfunction was still present in diabetic animals at day 28. This easily reproducible model provides clear insight into the evolution over time of the impact of ischemia on nerve and microvessel homeostasis in the setting of diabetes. © 2015 Wiley Periodicals, Inc.

The role of the inflammasome in cardiovascular diseases.

Inflammasome is a very important signaling platform sensing a variety of triggers of the innate immune system. Inflammasome promotes the production of important pro-inflammatory cytokines such as IL-1ß and IL-18. Tight control of inflammasome activity is, therefore, essential and occurs at multiple levels. The activation of inflammasome pathways is linked to the pathogenesis of various prevalent disorders including cardiovascular disease such as atherosclerosis, ischemic injury, cardiomyopathy, and Kawasaki disease. The study of the inflammasome in the cardiovascular system has led to the identification of important triggers and endogenous modulators, and to the exploration of new treatment strategies based on the inhibition of inflammasome activation or its end products, i.e., IL-1ß and IL-18. In summary, the discovery of the inflammasome has greatly advanced our understanding of how the innate immune system interferes with cardiovascular disease development and progression, and targeting inflammasome provides new avenues for the treatment and management of cardiovascular diseases.

MFGE8 inhibits inflammasome-induced IL-1ß production and limits postischemic cerebral injury.

Milk fat globule-EGF 8 (MFGE8) plays important, nonredundant roles in several biological processes, including apoptotic cell clearance, angiogenesis, and adaptive immunity. Several recent studies have reported a potential role for MFGE8 in regulation of the innate immune response; however, the precise mechanisms underlying this role are poorly understood. Here, we show that MFGE8 is an endogenous inhibitor of inflammasome-induced IL-1ß production. MFGE8 inhibited necrotic cell-induced and ATP-dependent IL-1ß production by macrophages through mediation of integrin ß(3) and P2X7 receptor interactions in primed cells. Itgb3 deficiency in macrophages abrogated the inhibitory effect of MFGE8 on ATP-induced IL-1ß production. In a setting of postischemic cerebral injury in mice, MFGE8 deficiency was associated with enhanced IL-1ß production and larger infarct size; the latter was abolished after treatment with IL-1 receptor antagonist. MFGE8 supplementation significantly dampened caspase-1 activation and IL-1ß production and reduced infarct size in wild-type mice, but did not limit cerebral necrosis in Il1b-, Itgb3-, or P2rx7-deficient animals. In conclusion, we demonstrated that MFGE8 regulates innate immunity through inhibition of inflammasome-induced IL-1ß production.

Glatiramer Acetate administration does not reduce damage after cerebral ischemia in mice.

Inflammation plays a key role in ischemic stroke pathophysiology: microglial/macrophage cells and type-1 helper cells (Th1) seem deleterious, while type-2 helper cells (Th2) and regulatory T cells (Treg) seem protective. CD4 Th0 differentiation is modulated by microglial cytokine secretion. Glatiramer Acetate (GA) is an immunomodulatory drug that has been approved for the treatment of human multiple sclerosis by means of a number of mechanisms: reduced microglial activation and pro-inflammatory cytokine production, Th0 differentiation shifting from Th2 to Th2 and Treg with anti-inflammatory cytokine production and increased neurogenesis. We induced permanent (pMCAo) or transient middle cerebral artery occlusion (tMCAo) and GA (2 mg) or vehicle was injected subcutaneously immediately after cerebral ischemia. Mice were sacrificed at D3 to measure neurological deficit, infarct volume, microglial cell density and qPCR of TNFα and IL-1ß (pro-inflammatory microglial cytokines), IFNγ (Th2 cytokine), IL-4 (Th2 cytokine), TGFß and IL-10 (Treg cytokines), and at D7 to evaluate neurological deficit, infarct volume and neurogenesis assessment. We showed that in GA-treated pMCAo mice, infarct volume, microglial cell density and cytokine secretion were not significantly modified at D3, while neurogenesis was enhanced at D7 without significant infarct volume reduction. In GA-treated tMCAo mice, microglial pro-inflammatory cytokines IL-1ß and TNFα were significantly decreased without modification of microglial/macrophage cell density, cytokine secretion, neurological deficit or infarct volume at D3, or modification of neurological deficit, neurogenesis or infarct volume at D7. In conclusion, Glatiramer Acetate administered after cerebral ischemia does not reduce infarct volume or improve neurological deficit in mice despite a significant increase in neurogenesis in pMCAo and a microglial pro-inflammatory cytokine reduction in tMCAo.

Muscle magnetic resonance imaging sensitivity does not decrease in chronic, mild, or proximal lower limb neuropathies.

INTRODUCTION: Muscle magnetic resonance imaging (MRI) is an innovative tool for exploring focal neuropathies. However, its usefulness in mild, proximal, or chronic lesions, when electromyography (EMG), the current "gold standard" sensitivity is inadequate, has yet to be studied. METHODS: Clinical, MRI, and EMG examinations were performed in 113 muscles of 17 consecutive patients with clinically diagnosed lower limb focal neuropathies. The sensitivity and specificity of MRI and EMG were evaluated in relation to disease duration, severity, and anatomical location. RESULTS: Muscle MRI was highly sensitive for the detection of denervated muscle, and, unlike EMG, its sensitivity did not decrease regardless of the anatomical location, duration, or severity of the neuropathy. Five MRI false positives were noted, including three in the thigh muscles. CONCLUSIONS: Muscle MRI is an alternative tool to EMG in proximal, mild, or chronic clinical diagnoses of lower limb focal neuropathies. However, it also seems prone to false-positive results, particularly in proximal muscles.

Neuroblast survival depends on mature vascular network formation after mouse stroke: role of endothelial and smooth muscle progenitor cell co-administration.

Pro-angiogenic cell-based therapies constitute an interesting and attractive approach to enhancing post-stroke neurogenesis and decreasing neurological deficit. However, most new stroke-induced neurons die during the first few weeks after ischemia, thus impairing total recovery. Although the neovascularization process involves different cell types and various growth factors, most cell therapy protocols are based on the biological effects of single-cell-type populations or on the administration of heterogeneous populations of progenitors, namely human cord blood-derived CD34(+) cells, with scarce vascular progenitor cells. Tight cooperation between endothelial cells and smooth muscle cells/pericytes is critical for the development of functional neovessels. We hypothesized that neuroblast survival in stroke brain depends on mature vascular network formation. In this study, we injected a combination of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs), isolated from human umbilical cord blood, into a murine model of permanent focal ischemia induced by middle cerebral artery occlusion. The co-administration of SMPCs and EPCs induced enhanced angiogenesis and vascular remodeling in the peri-infarct and infarct areas, where vessels exhibited a more mature phenotype. This activation of vessel growth resulted in the maintenance of neurogenesis and neuroblast migration to the peri-ischemic cortex. Our data suggest that a mature vascular network is essential for neuroblast survival after cerebral ischemia, and that co-administration of EPCs and SMPCs may constitute a novel therapeutic strategy for improving the treatment of stroke.

Dual action of NO synthases on blood flow and infarct volume consecutive to neonatal focal cerebral ischemia.

Research into neonatal ischemic brain damage is impeded by the lack of a complete understanding of the initial hemodynamic mechanisms resulting in a lesion, particularly that of NO-mediated vascular mechanisms. In a neonatal stroke rat model, we recently show that collateral recruitment contributes to infarct size variability. Non-specific and selective NO synthase (NOS) inhibition was evaluated on cerebral blood-flow changes and outcome in a P7 rat model of arterial occlusion (left middle cerebral artery electrocoagulation with 50 min occlusion of both common carotid arteries). Blood-flow changes were measured by using ultrasound imaging with sequential Doppler recordings in both internal carotid arteries and basilar trunk. Cortical perfusion was measured by using laser Doppler flowmetry. We showed that global NOS inhibition significantly reduced collateral support and cortical perfusion (collateral failure), and worsened the ischemic injury in both gender. Conversely, endothelial NOS inhibition increased blood-flows and aggravated volume lesion in males, whereas in females blood-flows did not change and infarct lesion was significantly reduced. These changes were associated with decreased phosphorylation of neuronal NOS at Ser(847) in males and increased phosphorylation in females at 24h, respectively. Neuronal NOS inhibition also increased blood-flows in males but not in females, and did not significantly change infarct volumes compared to their respective PBS-treated controls. In conclusion, both nNOS and eNOS appear to play a key role in modulating arterial blood flow during ischemia mainly in male pups with subsequent modifications in infarct lesion.

Transcranial duplex sonography for monitoring circle of Willis artery occlusion in a rat embolic stroke model.

BACKGROUND: Hemodynamic monitoring of circle of Willis arteries during clot embolization in experimental embolic stroke models (ES) is of major importance to assess their reproducibility and to test new recanalization strategies. We sought to assess the potential of transcranial duplex sonography (TDS) in comparison with laser Doppler flowmetry (LDF) to monitor the time of recanalization after occlusion of the right distal intracranial internal carotid artery (rICA) and to predict infarct volume at 24h after ischemia. METHODS: Twenty nine male Sprague-Dawley rats were submitted to ES. Right and left ICA, posterior cerebral arteries, and basilar trunk (BT) were monitored by TDS and regional cerebral blood flow was monitored by LDF at baseline, 1, 4 and 24h after the ES onset. Infarct volume was measured at 24h after stroke. RESULTS: Among the 25 rats, 3 died during surgery, 5 did not show any significant brain infarct (failure of the stroke model) and absence of occlusion of the rICA was detected in all 5 rats by TDS at 1h whereas only 4 of 5 were detected by LDF. Among the remaining 17 rats, the recanalization time of the occluded rICA assessed by TDS correlated with the infarct volume at 24h, r=0.70, P=0.0013 by contrast to results with LDF, r=0.11, P=0.55. CONCLUSION: Although both TDS and LDF can be used for in vivo assessment of occlusion in a rat ES model, TDS was more reliable as it allowed more accurate monitoring of arterial recanalization, and prediction of infarct volume.

Impact of intracranial blood-flow redistribution on stroke size during ischemia-reperfusion in 7-day-old rats.

We evaluated color-coded pulsed Doppler ultrasound imaging for the assessment of intracranial blood flow in two models of cerebral ischemia in 7-day-old (P7) rats. Blood-flow velocities (BFVs) were measured in the internal carotid arteries and basilar trunk upstream from the circle of Willis, and in the posterior cerebral arteries downstream (1) before, (2) during left middle cerebral artery electrocoagulation and 50 min-transient either one (I/R-1) or both (I/R-2) common carotid (CCA) arteries occlusion, and (3) after release of CCA(s) occlusion. At 48 h after ischemia 41-48% (I/R-1 model) and 24% (I/R-2 model) of rats did not present a lesion. Those rats displayed increased mean BFV in both right internal carotid artery and basilar trunk in I/R-1 model, and increased mean BFV in the basilar trunk (BT) in I/R-2 model. In contrast, no significant changes in mean BFV were observed in lesioned rats. Furthermore, mean BFV in the BT was inversely correlated to the size of the lesion (r² = 0.75, p<0.0001) in the I/R-2 model. Thus, we demonstrated the protective role of collateral support in P7 rodents. Ultrasound imaging can evidence the establishment or not of the cerebral collateral recruitment, leading to the presence or absence of a lesion. This novel approach should greatly help preclinical studies to reduce animal variability.

Triple-stimulation technique in multifocal neuropathy with conduction block.

In patients with multifocal neuropathy with conduction block (CB), CBs located between the root and Erb's point are not detected in nerve conduction studies. We therefore examined whether the triple-stimulation technique (TST) might provide a useful means of detecting CB proximal to Erb's point. Clinical assessments, extensive nerve conduction studies (NCS), conventional transcranial magnetic stimulation, and TST were performed on 10 patients with multifocal motor neuropathy with CB (MMNCB) and 6 patients with Lewis-Sumner syndrome. Conduction blocks located proximal to Erb's point were detected in 9 patients. Of the CBs, 58% were associated with muscle weakness. The use of TST to detect proximal CB improved the sensitivity of the American Association of Neuromuscular and Electrodiagnostic Medicine criteria for definite or probable MMNCB from 60% to 90%. Thus, the TST is a useful means for detection of proximal CB and gives NCS considerable additional diagnostic power.