Cornea graft endothelial cells undergo apoptosis by way of an alternate (caspase-independent) pathway.

PURPOSE: To look for apoptosis pathways involved in corneal endothelial cell death during acute graft rejection and to evaluate the potential role of nitric oxide in this process. MATERIALS AND METHODS: Corneal buttons from Brown-Norway rats were transplanted into Lewis rat corneas. At different time intervals after transplantation, apoptosis was assessed by diamino-2-phenylindol staining and annexin-V binding on flat-mount corneas, and by terminal transferase dUTP nick end labeling (TUNEL), caspase-3 dependent and leukocyte elastase inhibitor (LEI)/LDNase II caspase-independent pathways on sections. Inducible nitric oxide synthase (NOS-II) expression and the presence of nitrotyrosine were assayed by immunohistochemistry. RESULTS: Graft endothelial cells demonstrated nuclear fragmentation and LEI nuclear translocation, annexin-V binding, and membranes bleb formation. Apoptosis associated with caspase-3 activity or TUNEL-positive reaction was not observed at any time either in the graft or in the recipient corneal endothelial cells. During 14 days posttransplantation, the recipient corneal endothelial cells remained unaltered and their number unchanged in all studied corneas. NOS-II was expressed in infiltrating cells present within the graft. This expression was closely associated with the presence of nitrotyrosine in endothelial and infiltrating cells. CONCLUSION: During the time course of corneal graft rejection, graft endothelial cells undergo apoptosis. Apoptosis is caspase 3 independent and TUNEL negative and is, probably, carried out by an alternative pathway driven by an LEI/L-Dnase II. Peroxynitrite formation may be an additional mechanism for cell toxicity and programmed cell death of the graft endothelial cells during the rejection process in this model.

PAX6 mutations in Egyptian families with aniridia

Methods Ten patients with aniridia from 3 families of Egyptian origin underwent full ophthalmologic, general and neurological examination, and blood drawing. Cerebral MRI was performed in the index case of each family. Genomic DNA was prepared from venous leukocytes and direct sequencing of all the exons and intron-exon junctions of the PAX6 gene was performed after PCR amplification. Results Common features observed in the three families included absence of iris tissue, corneal pannus with different degrees of severity and foveal hypoplasia with severely reduced visual acuity. In families 2 and 3, additional findings such as lens dislocation, lens opacities or polar cataract and glaucoma were observed. We identified two novel (c.170-174delTGGGC [p.L57fs17] and c.475delC [p.R159fs47]) and one known (c.718C>T) PAX6 mutations in the affected members of the 3 families. Systemic and neurological examination was normal in all ten affected patients. Cerebral MRI showed absence of the pineal gland in all three index patients. Severe hypoplasia of the brain anterior commissure was associated to the p.L57fs17mutation, absence of the posterior commissure to both p.R159fs47 and p.R240X, and optic chiasma atrophy and almost complete agenesis of the corpus callosum to p.R240X. Conclusions We identified two novel PAX6 mutations in families with severe aniridia from Northern Egypt, an ethnic group which is not well studied. In addition to common phenotype of aniridia and despite normal neurological examination, absence of the pineal gland was observed in all 3 index patients. The heterogeneity of brain anomalies related to PAX6 mutations is underexplored and is highlighted in this study.

Patient adherence and the choice of antihypertensive drugs: focus on lercanidipine.

Despite the development of many effective antihypertensive drugs, target blood pressures are reached in only a minority of patients in clinical practice. Poor adherence to drug therapy and the occurrence of side effects are among the main reasons commonly reported by patients and physicians to explain the poor results of actual antihypertensive therapies. The development of new effective antihypertensive agents with an improved tolerability profile might help to partly overcome these problems. Lercanidipine is an effective dihydropyridine calcium channel blocker of the third generation characterized by a long half-life and its lipophylicity. In contrast to first-generation dihydropyridines, lercanidipine does not induce reflex tachycardia and induces peripheral edema with a lower incidence. Recent data suggest that in addition to lowering blood pressure, lercanidipine might have some renal protective properties. In this review we shall discuss the problems of drug adherence in the management of hypertension with a special emphasis on lercanidipine.

Kératocône bilatéral évolué [Bilateral keratoconus: a case report].

Among corneal dystrophies, the keratoconus is one of the most frequently observed among young adults. A clinico pathological case is reported in a 13-year-old-girl of African origin. The diagnosis of bilateral keratoconus was established based on the obvious changes of the corneal curvature and thickness. After an unsuccessful attempt to improve vision with contact lenses, a keratoplasty was finally performed on one side to remove the pathological cornea. Its histopathological study found the characteristic changes of keratoconus: breaks of Bowman's layer and corneal thinning.

The role of bradykinin B(1) and B(2) receptors for secondary brain damage after traumatic brain injury in mice.

Inflammatory mechanisms are known to contribute to the pathophysiology of traumatic brain injury (TBI). Since bradykinin is one of the first mediators activated during inflammation, we investigated the role of bradykinin and its receptors in posttraumatic secondary brain damage. We subjected wild-type (WT), B(1)-, and B(2)-receptor-knockout mice to controlled cortical impact (CCI) and analyzed tissue bradykinin as well as kinin receptor mRNA and protein expression up to 48 h thereafter. Brain edema, contusion volume, and functional outcome were assessed 24 h and 7 days after CCI. Tissue bradykinin was maximally increased 2 h after trauma (P<0.01 versus sham). Kinin B(1) receptor mRNA was upregulated up to four-fold 24 h after CCI. Immunohistochemistry showed that B(1) and B(2) receptors were expressed in the brain and were significantly upregulated in the traumatic penumbra 1 to 24 h after CCI. B(2)R(-/-) mice had significantly less brain edema (-51% versus WT, 24 h; P<0.001), smaller contusion volumes ( approximately 50% versus WT 24 h and 7 d after CCI; P<0.05), and better functional outcome 7 days after TBI as compared with WT mice (P<0.05). The present results show that bradykinin and its B(2) receptors play a causal role for brain edema formation and cell death after TBI.

Ocular antisense oligonucleotide delivery by cationic nanoemulsion for improved treatment of ocular neovascularization: an in-vivo study in rats and mice.

The efficacy of an antisense oligonucleotide (ODN17) cationic nanoemulsion directed at VEGF-R2 to reduce neovascularization was evaluated using rat corneal neovascularization and retinopathy of prematurity (ROP) mouse models. Application of saline solution or scrambled ODN17 solution on eyes of rats led to the highest extent of corneal neovascularization. The groups treated with blank nanoemulsion or scrambled ODN17 nanoemulsion showed moderate inhibition in corneal neovascularization with no significant difference with the saline and scrambled ODN17 control solution groups, while the groups treated with ODN17 solution or Avastin® (positive ODN17 control) clearly elicited marked significant inhibition in corneal neovascularization confirming the results reported in the literature. The highest significant corneal neovascularization inhibition efficiency was noted in the groups treated with ODN17 nanoemulsion (topical and subconjunctivally). However, in the ROP mouse model, the ODN17 in PBS induced a 34% inhibition of retinal neovascularization when compared to the aqueous-vehicle-injected eyes. A significantly higher inhibition of vitreal neovascularization (64%) was observed in the group of eyes treated with ODN17 nanoemulsion. No difference in extent of neovascularization was observed between blank nanoemulsion, scrambled ODN17 nanoemulsion, vehicle or non-treated eyes. The overall results indicate that cationic nanoemulsion can be considered a promising potential ocular delivery system and an effective therapeutic tool of high clinical significance in the prevention and forthcoming treatment of ocular neovascular diseases.

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.

Pathologies mimicking deep venous thrombosis of the legs : pictorial review of US findings : P27

Purpose: Precise diagnosis of DVT of the legs is a challenging problem, not only in front of suspicion of PE, but also in all status of leg pain, warmth and swelling. Clinical diagnosis has a low accuracy and further investigations are mandatory in order to diagnose DVT. Amongst the possible investigations, US has a high specificity and a good NPV. However, many pathologies unrelated to the veins may mimic the signs and symptoms of DVT and have to be recognized in order to make the correct diagnosis. The purpose of this paper is to review the results of the US investigations of the legs performed in our Department during the last three years for a suspicion of DVT and describe alternative diagnoses mimicking DVT. Methods and materials: Through a RIS-based search, we retrospectively reviewed all the cases of US of the legs performed in our Department between January 2006 and December 2008 for a suspicion of DVT. We selected the cases of positive findings unrelated to the veins and illustrated these findings with characteristic images. Results: 419 US of the legs were performed between December 2006 and December 2008 for a suspicion of DVT. Among these, 75 were positive for DVT, and 79 for alternative diagnosis. The most common alternative diagnosis was edema of the legs (31%), followed by hematoma (23%). Other findings were Baker cysts (13%), cellulitis (10%) and lymphoceles (5%). Rare diagnoses were arterio-venous malformations, pseudoaneurysms, pelvic masses, necrosing fasciitis, intramuscular abscesses, subcutaneous seromas, sarcoma and ganglion cysts. Conclusion: A greater knowledge of the US appearance of the pathologies mimicking DVT may help to make the correct diagnosis, avoiding further expensive investigations or inappropriate anticoagulant therapy.

MRI monitoring of focal cerebral ischemia in peroxisome proliferator-activated receptor (PPAR)-deficient mice.

Peroxisome proliferator-activated receptors (PPARs) are a potential target for neuroprotection in focal ischemic stroke. These nuclear receptors have major effects in lipid metabolism, but they are also involved in inflammatory processes. Three PPAR isotypes have been identified: alpha, beta (or delta) and gamma. The development of PPAR transgenic mice offers a promising tool for prospective therapeutic studies. This study used MRI to assess the role of PPARalpha and PPARbeta in the development of stroke. Permanent middle cerebral artery occlusion induced focal ischemia in wild-type, PPARalpha-null mice and PPARbeta-null mice. T(2)-weighted MRI was performed with a 7 T MRI scan on day 0, 1, 3, 7 and 14 to monitor lesion growth in the various genotypes. General Linear Model statistical analysis found a significant difference in lesion volume between wild-type and PPAR-null mice for both alpha and beta isotypes. These data validate high-resolution MRI for monitoring cerebral ischemic lesions, and confirm the neuroprotective role of PPARalpha and PPARbeta in the brain.

Importance of ENaC-mediated sodium transport in alveolar fluid clearance using genetically-engineered mice.

The lung possesses specific transport systems that intra- and extracellularly maintain salt and fluid balance necessary for its function. At birth, the lungs rapidly transform into a fluid (Na(+))-absorbing organ to enable efficient gas exchange. Alveolar fluid clearance, which mainly depends on sodium transport in alveolar epithelial cells, is an important mechanism by which excess water in the alveoli is reabsorbed during the resolution of pulmonary edema. In this review, we will focus and summarize on the role of ENaC in alveolar lung liquid clearance and discuss recent data from mouse models with altered activity of epithelial sodium channel function in the lung, and more specifically in alveolar fluid clearance. Recent data studying mice with hyperactivity of ENaC or mice with reduced ENaC activity clearly illustrate the impaired lung fluid clearance in these adult mice. Further understanding of the physiological role of ENaC and its regulatory proteins implicated in salt and water balance in the alveolar cells may therefore help to develop new therapeutic strategies to improve gas exchange in pulmonary edema.

Pulmonary and systemic vascular dysfunction in young offspring of mothers with preeclampsia.

BACKGROUND: Adverse events in utero may predispose to cardiovascular disease in adulthood. The underlying mechanisms are unknown. During preeclampsia, vasculotoxic factors are released into the maternal circulation by the diseased placenta. We speculated that these factors pass the placental barrier and leave a defect in the circulation of the offspring that predisposes to a pathological response later in life. The hypoxia associated with high-altitude exposure is expected to facilitate the detection of this problem. METHODS AND RESULTS: We assessed pulmonary artery pressure (by Doppler echocardiography) and flow-mediated dilation of the brachial artery in 48 offspring of women with preeclampsia and 90 offspring of women with normal pregnancies born and permanently living at the same high-altitude location (3600 m). Pulmonary artery pressure was roughly 30% higher (mean+/-SD, 32.1+/-5.6 versus 25.3+/-4.7 mm Hg; P<0.001) and flow-mediated dilation was 30% smaller (6.3+/-1.2% versus 8.3+/-1.4%; P<0.0001) in offspring of mothers with preeclampsia than in control subjects. A strong inverse relationship existed between flow-mediated dilation and pulmonary artery pressure (r=-0.61, P<0.001). The vascular dysfunction was related to preeclampsia itself because siblings of offspring of mothers with preeclampsia who were born after a normal pregnancy had normal vascular function. Augmented oxidative stress may represent an underlying mechanism because thiobarbituric acid-reactive substances plasma concentration was increased in offspring of mothers with preeclampsia. CONCLUSIONS: Preeclampsia leaves a persistent defect in the systemic and the pulmonary circulation of the offspring. This defect predisposes to exaggerated hypoxic pulmonary hypertension already during childhood and may contribute to premature cardiovascular disease in the systemic circulation later in life.

Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice.

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.

Nitric oxide synthase (NOS) inhibition for one week improves renal sodium and water excretion in cirrhotic rats with ascites

Normalization of the increased vascular nitric oxide (NO) generation with low doses of NG-nitro-L-arginine methyl ester (L-NAME) corrects the hemodynamic abnormalities of cirrhotic rats with ascites. We have undertaken this study to investigate the effect of the normalization of vascular NO production, as estimated by aortic cyclic guanosine monophosphate (cGMP) concentration and endothelial nitric oxide synthase (eNOS) protein expression in the aorta and mesenteric artery, on sodium and water excretion. Rats with carbon tetrachloride-induced cirrhosis and ascites were investigated using balance studies. The cirrhotic rats were separated into two groups, one receiving 0.5 mg/kg per day of L-NAME (CIR-NAME) during 7 d, whereas the other group (CIR) was administrated the same volume of vehicle. Two other groups of rats were used as controls, one group treated with L-NAME and another group receiving the same volume of vehicle. Sodium and water excretion was measured on days 0 and 7. On day 8, blood samples were collected for electrolyte and hormone measurements, and aorta and mesenteric arteries were harvested for cGMP determination and nitric oxide synthase (NOS) immunoblotting. Aortic cGMP and eNOS protein expression in the aorta and mesenteric artery were increased in CIR as compared with CIR-NAME. Both cirrhotic groups had a similar decrease in sodium excretion on day 0 (0.7 versus 0.6 mmol per day, NS) and a positive sodium balance (+0.9 versus +1.2 mmol per day, NS). On day 7, CIR-NAME rats had an increase in sodium excretion as compared with the CIR rats (sodium excretion: 2.4 versus 0.7 mmol per day, P < 0.001) and a negative sodium balance (-0.5 versus +0.8 mmol per day, P < 0.001). The excretion of a water load was also increased after L-NAME administration (from 28+/-5% to 65+/-7, P < 0.05). Plasma renin activity, aldosterone and arginine vasopressin were also significantly decreased in the CIR-NAME, as compared with the CIR rats. The results thus indicate that normalization of aortic cGMP and eNOS protein expression in vascular tissue is associated with increased sodium and water excretion in cirrhotic rats with ascites.

Neuromonitoring after major neurosurgical procedures.

Postoperative care of major neurosurgical procedures is aimed at the prevention, detection and treatment of secondary brain injury. This consists of a series of pathological events (i.e. brain edema and intracranial hypertension, cerebral hypoxia/ischemia, brain energy dysfunction, non-convulsive seizures) that occur early after the initial insult and surgical intervention and may add further burden to primary brain injury and thus impact functional recovery. Management of secondary brain injury requires specialized neuroscience intensive care units (ICU) and continuous advanced monitoring of brain physiology. Monitoring of intracranial pressure (ICP) is a mainstay of care and is recommended by international guidelines. However, ICP monitoring alone may be insufficient to detect all episodes of secondary brain insults. Additional invasive (i.e. brain tissue PO2, cerebral microdialysis, regional cerebral blood flow) and non-invasive (i.e. transcranial doppler, near-infrared spectroscopy, EEG) brain monitoring devices might complement ICP monitoring and help clinicians to target therapeutic interventions (e.g. management of cerebral perfusion pressure, blood transfusion, glucose control) to patient-specific pathophysiology. Several independent studies demonstrate such multimodal approach may optimize patient care after major neurosurgical procedures. The aim of this review is to evaluate some of the available monitoring systems and summarize recent important data showing the clinical utility of multimodal neuromonitoring for the management of main acute neurosurgical conditions, including traumatic brain injury, subarachnoid hemorrhage and stroke.