Deletion of aquaporin-4 improves capillary blood flow distribution in brain edema.

Brain edema is a feared complication to disorders and insults affecting the brain. It can be fatal if the increase in intracranial pressure is sufficiently large to cause brain herniation. Moreover, accruing evidence suggests that even slight elevations of intracranial pressure have adverse effects, for instance on brain perfusion. The water channel aquaporin-4 (AQP4), densely expressed in perivascular astrocytic endfeet, plays a key role in brain edema formation. Using two-photon microscopy, we have studied AQP4-mediated swelling of astrocytes affects capillary blood flow and intracranial pressure (ICP) in unanesthetized mice using a mild brain edema model. We found improved regulation of capillary blood flow in mice devoid of AQP4, independently of the severity of ICP increase. Furthermore, we found brisk AQP4-dependent astrocytic Ca2+ signals in perivascular endfeet during edema that may play a role in the perturbed capillary blood flow dynamics. The study suggests that astrocytic endfoot swelling and pathological signaling disrupts microvascular flow regulation during brain edema formation.

Anesthesia-related brain microstructure modulations detected by diffusion magnetic resonance imaging.

Recent studies have shown significant changes to brain microstructure during sleep and anesthesia. In vivo optical microscopy and magnetic resonance imaging (MRI) studies have attributed these changes to anesthesia and sleep-related modulation of the brain's extracellular space (ECS). Isoflurane anesthesia is widely used in preclinical diffusion MRI (dMRI) and it is therefore important to investigate if the brain's microstructure is affected by anesthesia to an extent detectable with dMRI. Here, we employ diffusion kurtosis imaging (DKI) to assess brain microstructure in the awake and anesthetized mouse brain (n = 22). We find both mean diffusivity (MD) and mean kurtosis (MK) to be significantly decreased in the anesthetized mouse brain compared with the awake state (p < 0.001 for both). This effect is observed in both gray matter and white matter. To further investigate the time course of these changes we introduce a method for time-resolved fast DKI. With this, we show the time course of the microstructural alterations in mice (n = 5) as they transition between states in an awake-anesthesia-awake paradigm. We find that the decrease in MD and MK occurs rapidly after delivery of gas isoflurane anesthesia and that values normalize only slowly when the animals return to the awake state. Finally, time-resolved fast DKI is employed in an experimental mouse model of brain edema (n = 4), where cell swelling causes the ECS volume to decrease. Our results show that isoflurane affects DKI parameters and metrics of brain microstructure and point to isoflurane causing a reduction in the ECS volume. The demonstrated DKI methods are suitable for in-bore perturbation studies, for example, for investigating microstructural modulations related to sleep/wake-dependent functions of the glymphatic system. Importantly, our study shows an effect of isoflurane anesthesia on rodent brain microstructure that has broad relevance to preclinical dMRI.

The Probe for Renal Organic Cation Secretion (4-Dimethylaminostyryl)-N-Methylpyridinium (ASP+)) Shows Amplified Fluorescence by Binding to Albumin and Is Accumulated In Vivo.

Accumulation of uremic toxins may lead to the life-threatening condition "uremic syndrome" in patients with advanced chronic kidney disease (CKD) requiring renal replacement therapy. Clinical evaluation of proximal tubular secretion of organic cations (OC), of which some are uremic toxins, is desired, but difficult. The biomedical knowledge on OC secretion and cellular transport partly relies on studies using the fluorescent tracer 4-dimethylaminostyryl)-N-methylpyridinium (ASP+), which has been used in many studies of renal excretion mechanisms of organic ions and which could be a candidate as a PET tracer. This study is aimed at expanding the knowledge of the tracer characteristics of ASP+ by recording the distribution and intensity of ASP+ signals in vivo both by fluorescence and by positron emission tomography (PET) imaging and at investigating if the fluorescence signal of ASP+ is influenced by the presence of albumin. Two-photon in vivo microscopy of male Münich Wistar Frömter rats showed that a bolus injection of ASP+ conferred a fluorescence signal to the blood plasma lasting for about 30 minutes. In the renal proximal tubule, the bolus resulted in a complex pattern of fluorescence including a rapid and strong transient signal at the brush border, a very low signal in the luminal fluid, and a slow transient intracellular signal. PET imaging using 11C-labelled ASP+ showed accumulation in the liver, heart, and kidney. Fluorescence emission spectra recorded in vitro of ASP+ alone and in the presence of albumin using both 1-photon excitation and two-photon excitation showed that albumin strongly enhance the emission from ASP+ and induce a shift of the emission maximum from 600 to 570 nm. Conclusion. The renal pattern of fluorescence observed from ASP+ in vivo is likely affected by the local concentration of albumin, and quantification of ASP+ fluorescent signals in vivo cannot be directly translated to ASP+ concentrations.

Is it possible to reduce recurrences after Altemeier's procedure for complete rectal prolapse? Twenty-year experience in 130 consecutive patients.

PURPOSE: In the attempt to understand the reasons for and to find a solution to the high recurrence rate after perineal surgery for complete rectal prolapse, we retrospectively analysed the long-term results of Altemeier's procedure alone, or associated with Trans-Obturator Colonic Suspension (TOCS) in a large series of patients with a median interval of 84 months (range 6-258). METHODS: Medical records of 110 patients undergoing Altemeier with levatorplasty (group 1) and 20 patients submitted to the same procedure associated with TOCS (group 2) for newly diagnosed complete rectal prolapse were reviewed. All patients had been recruited after preoperative clinical examination, SF-36 quality of life, continence score and colonoscopy. RESULTS: Mortality was nil. The overall complication and the recurrence rates were 12.3%, and 15.0% (P= 0.769) and 24.6% and 5.0% (P=0.067) in group 1 and 2, respectively. Twelve patients of group 1 with a recurrence were submitted to a redo-Altemeier, 8 to a redo-Altemeier associated with TOCS, and 6 associated with an anterior coloplasty with a mesh. The only patient of group 2 with a recurrence was submitted to a Hartmann's operation. Preoperative vs postoperative mean (SD) continence score was 15.8 (3.1) and 15.6 (3.3) versus 4.1 (1.8) and 3.9 (1.9) in group 1 and 2, respectively (P < 0.001). All parameters of SF-36 improved after surgery (P<0.01) and no differences between the 2 groups were found CONCLUSIONS: Long-term results confirmed the safety and effectiveness of Altemeier's procedure for the treatment of complete rectal prolapse, with the limit of a non-negligible incidence of anastomotic complications and recurrences. The combination of Altemeier with TOCS showed a positive trend to a reduction of the recurrence rate, not worsening morbidity and outcomes.

The evidence for the physiological effects of lactate on the cerebral microcirculation: a systematic review.

Lactate's role in the brain is understood as a contributor to brain energy metabolism, but it may also regulate the cerebral microcirculation. The purpose of this systematic review was to evaluate evidence of lactate as a physiological effector within the normal cerebral microcirculation in reports ranging from in vitro experiments to in vivo studies in animals and humans. Following pre-registration of a review protocol, we systematically searched the PubMed, EMBASE, and Cochrane databases for literature covering themes of 'lactate', 'the brain', and 'microcirculation'. Abstracts were screened, and data extracted independently by two individuals. We excluded studies evaluating lactate in disease models. Twenty-eight papers were identified, 18 of which were in vivo animal experiments (65%), four on human studies (14%), and six on in vitro or ex vivo experiments (21%). Approximately half of the papers identified lactate as an augmenter of the hyperemic response to functional activation by a visual stimulus or as an instigator of hyperemia in a dose-dependent manner, without external stimulation. The mechanisms are likely to be coupled to NAD+ /NADH redox state influencing the production of nitric oxide. Unfortunately, only 38% of these studies demonstrated any control for bias, which makes reliable generalizations of the conclusions insecure. This systematic review identifies that lactate may act as a dose-dependent regulator of cerebral microcirculation by augmenting the hyperemic response to functional activation below 5 mmol/kg, and by initiating a hyperemic response above 5 mmol/kg. OPEN SCIENCE BADGES: This article has received a badge for *Pre-registration* because it made the data publicly available. The data can be accessed at www.radboudumc.nl/getmedia/53625326-d1df-432c-980f-27c7c80d1a90/THollyer_lactate_protocol.aspx. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.

Longitudinal tracking of acute kidney injury reveals injury propagation along the nephron.

Acute kidney injury (AKI) is an important risk factor for chronic kidney disease (CKD), but the underlying mechanisms of failed tubule repair and AKI-CKD transition are incompletely understood. In this study, we aimed for dynamic tracking of tubule injury and remodeling to understand if focal injury upon AKI may spread over time. Here, we present a model of AKI, in which we rendered only half of the kidney ischemic. Using serial intravital 2-photon microscopy and genetic identification of cycling cells, we tracked dynamic tissue remodeling in post- and non-ischemic kidney regions simultaneously and over 3 weeks. Spatial and temporal analysis of cycling cells relative to initial necrotic cell death demonstrated pronounced injury propagation and expansion into non-necrotic tissue regions, which predicted tubule atrophy with epithelial VCAM1 expression. In summary, our longitudinal analyses of tubule injury, remodeling, and fate provide important insights into AKI pathology.

A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis.

Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease.

Quantification of Capillary Perfusion in an Animal Model of Acute Intracranial Hypertension.

Intracranial hypertension (IH) is a common feature of many pathologies, including brain edema. In the brain, the extended network of capillaries ensures blood flow to meet local metabolic demands. Capillary circulation may be severely affected by IH, but no studies have quantified the effect of intracranial pressure (ICP) and cerebral perfusion pressure (CPP) on capillary perfusion during the development of brain edema. We used optical coherence tomography angiography to quantify relative changes of fractional perfused volume (FPV) in cortical capillaries and simultaneously monitored ICP and blood pressure (BP) in anesthetized male C57Bl/6NTac mice during development of brain edema induced by water intoxication (WI) within 30 min. WI induced severe IH and brain herniation. ICP and CPP reached 90.2 mm Hg and 38.4 mm Hg, respectively. FPV was significantly affected already at normal ICP (ICP <15 mm Hg, slope ≈ -1.46, p < 0.001) and, at the onset of IH (ICP = 20-22 mm Hg), FPV was 17.9 ± 13.3% lower than baseline. A decreasing trend was observed until the ICP peak (Δ%FPV = -43.6 ± 19.2%). In the ICP range of 7-42 mm Hg, relative changes in FPV were significantly correlated with ICP, BP, and CPP (p < 0.001), with ICP and CPP being the best predictors. In conclusion, elevated ICP induces a gradual collapse of the cerebral microvasculature, which is initiated before the clinical threshold of IH. In summary, the estimate of capillary perfusion might be essential in patients with IH to assess the state of the brain microcirculation and to improve the availability of oxygen and nutrients to the brain.

A Minimally Invasive Technique for the 1-Stage Treatment of Complex Pelvic Floor Diseases: Laparoscopic-Pelvic Organ Prolapse Suspension.

OBJECTIVE: The aim of this prospective study was to assess the safety and effectiveness of a new single laparoscopic operation devised to relieve obstructed defecation, gynecologic and urinary symptoms in a large series of female patients with multiorgan pelvic prolapse. METHODS: We submitted 384 female patients to laparoscopic pelvic organ prolapse suspension operation, a new technique based on suspension of the middle pelvic compartment, by using a polypropylene mesh and followed up 368 of them, with defecography performed 12 months after surgery and a standardized protocol. RESULTS: The 368 patients were followed-up for 36.3 (±4.4) months, Recurrence rate was 4.9% for obstructed defecation syndrome and 3.3% for stress urinary incontinence. Complication rate was 2.9%. The mean period of daily activity resumption was 16.3 days (±4.8 days). Anorectal and urogynecologic symptoms and scores significantly improved after the operation (P < 0.001), with no worsening of anal continence. Incidence of postoperative fecal urgency was 0%. Postoperative defecography showed a significant (P < 0.001) improvement of all parameters in 315 patients (82%). Short Form 36 Health Survey score significantly improved after the operation (P < 0.01). An excellent/good overall Satisfaction Index was reported by 78.0% of patients. CONCLUSIONS: In our experience the Laparoscopic-Pelvic Organ Prolapse Suspension seems to be safe and effective as a 1-stage treatment of associated pelvic floor diseases. Randomized studies with an appropriate control group and longer follow-up are now needed to assess the effectiveness of this promising technique.

A Porthole over AKI: Cell Dynamics in Damage and Repair.

Acute kidney injury (AKI) is associated with an increased risk of CKD. Injury-induced multifaceted renal cell-to-cell crosstalk can either lead to successful self-repair or chronic fibrosis and inflammation. In this mini-review, we will discuss critical renal cell types acting as victims or executioners in AKI pathology and introduce intravital imaging as a powerful technique to further dissect these cell-to-cell interactions.

A new experimental mouse model of water intoxication with sustained increased intracranial pressure and mild hyponatremia without side effects of antidiuretics.

The most used experimental mouse model of hyponatremia and elevated intracranial pressure (ICP) is intraperitoneal injection of water in combination with antidiuretics. This model of water intoxication (WI) results in extreme pathological changes and death within 1 h. To improve preclinical studies of the pathophysiology of elevated ICP, we characterized diuresis, cardiovascular parameters, blood ionogram and effects of antidiuretics in this model. We subsequently developed a new mouse model with mild hyponatremia and sustained increased ICP. To investigate the classical protocol (severe WI), C57BL/6mice were anesthetized and received an intraperitoneal injection of 20% body weight of MilliQ water with or without 0.4 µg·kg-1 desmopressin acetate (dDAVP). Corresponding Sham groups were also studied. In the new WI protocol (mild WI), 10% body weight of a solution containing 6.5 mM NaHCO3, 1.125 mM KCl and 29.75 mM NaCl was intraperitoneally injected. By severe WI, ICP and mean arterial pressure increased until brain stem herniation occurred (23 ± 3 min after injection). The cardiovascular effects were accelerated by dDAVP. Severe WI induced a halt to urine production irrespective of the use of dDAVP. Following the new mild WI protocol, ICP also increased but was sustained at a pathologically high level without inducing herniation. Mean arterial pressure and urine production were not affected during mild WI. In conclusion, the new mild WI protocol is a superior experimental model to study the pathophysiological effects of elevated ICP induced by water intoxication.

The evolution of transanal surgery for obstructed defecation syndrome: Mid-term results from a randomized study comparing double TST 36 HV and Contour TRANSTAR staplers.

A randomized study was carried out to compare the mid-term outcome of transanal rectal resection with the CCS-30 TRANSTAR and two TST36 staplers in patients with obstructed defecation syndrome. After selection, patients were randomly assigned to 2 groups:104 underwent a TRANSTAR operation and 104 a transanal rectal resection with two TST36 staplers. Patients were followed up with clinical examination, and defecography. Cumulative complication rate was significantly higher in TRANSTAR operation (P = 0.019). All symptoms and defecographic parameters significantly improved (P < 0.001), without differences. Costs were significantly lower with double TST (P = 0.035). Recurrence rates were 6.2% in TRANSTAR group and 11.4% with double TST (P = 0.206). Two circular TST 36 staplers consent to obtain the same clinical and functional results than the CCS-30, with significantly lower complication rate and costs.

Anti-inflammatory activity of thymol: inhibitory effect on the release of human neutrophil elastase.

Elastase, a serine proteinase released by activated human neutrophils, can degrade a wide variety of biomacromolecules including elastin, and is considered a marker of inflammatory diseases. As the logical strategy to protect tissue is to inhibit excessive elastase activity, experimental and clinical researches have concentrated on trying to find efficient elastase inhibitors. As thymol, one of the major components of thyme oil with a phenolic structure, has been credited with a series of pharmacological properties, that include antimicrobial and antioxidant effects, the aim of this study was to explore whether it can also interfere with the release of elastase by human neutrophils stimulated with the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). After the neutrophils were incubated with increasing amounts of thymol (2.5, 5, 10, 20 microg/ml), elastase release was initiated by fMLP and measured using MeO-Suc-Ala-Ala-Pro-Val-MCA. The results showed that thymol inhibited fMLP-induced elastase release in a concentration-dependent manner, with the effects of 10 and 20 microg/ml being statistically significant. The behavior of cytosolic calcium mobilization revealed by fura-2 closely resembled that of elastase, thus suggesting that they may be related. The hydrophobic nature of thymol means that it can approach ion channel proteins through the lipid phase of the membrane, alter the local environment of calcium channels and thus inhibit capacitative calcium entry. In brief, thymol inactivates calcium channels machinery, thus triggering a corresponding reduction in elastase. The antibacterial and antimycotic activity of thymol is already well known, but our findings that it inhibits elastase extend our knowledge of the anti-inflammatory activity of this interesting molecule that is already credited with antioxidant activity. These two latter characteristics make thymol a molecule that can have helpful effects in controlling the inflammatory processes present in many infections.