Endothelial surface translocation of mitochondrial PDCE2 involves the non-canonical secretory autophagy pathway: Putative molecular target for radiation-guided drug delivery.

Radiation has been proposed as a priming agent to induce discriminatory luminal biomarkers for vascular targeting and drug delivery in disorders such as brain arteriovenous malformations and cancers. We previously observed ectopic expression of intracellular proteins such as mitochondrial PDCE2 on irradiated endothelium in animal models. In this study we examined the mechanism of PDCE2 trafficking in human endothelial cells to better understand its suitability as a vascular target. Ionizing radiation induced PDCE2 surface localization in association with accumulation of autophagosome markers (L3CB and p62) indicative of late-stage inhibition of autophagic flux. This effect was abolished in the presence of Rapamycin, an autophagy-inducer, but replicated in the presence of Bafilomycin A, an autophagy blocker. PDCE2 co-localized with lysosomal markers of the canonical degradative autophagy pathway in response to radiation but also with recycling endosomes and SNARE proteins responsible for autophagosome-plasma membrane fusion. These findings demonstrate that radiation-induced blockade of autophagic flux stimulates redirection of intracellular molecules such as PDCE2 to the cell surface via a non-canonical secretory autophagy pathway. Intracellular membrane proteins trafficked in this way could provide a unique pool of radiation biomarkers for therapeutic drug delivery.

Stable thrombus formation on irradiated microvascular endothelial cells under pulsatile flow: Pre-testing annexin V-thrombin conjugate for treatment of brain arteriovenous malformations.

BACKGROUND: Our goal is to develop a vascular targeting treatment for brain arteriovenous malformations (AVMs). Externalized phosphatidylserine has been established as a potential biomarker on the endothelium of irradiated AVM blood vessels. We hypothesize that phosphatidylserine could be selectively targeted after AVM radiosurgery with a ligand-directed vascular targeting agent to achieve localized thrombosis and rapid occlusion of pathological AVM vessels. OBJECTIVE: The study aim was to establish an in vitro parallel-plate flow chamber to test the efficacy of a pro-thrombotic conjugate targeting phosphatidylserine. METHODS: Conjugate was prepared by Lys-Lys cross-linking of thrombin with the phosphatidylserine-targeting ligand, annexin V. Cerebral microvascular endothelial cells were irradiated (5, 15, and 25 Gy) and after 1 or 3 days assembled in a parallel-plate flow chamber containing whole human blood and conjugate (1.25 or 2.5 µg/mL). Confocal microscopy was used to assess thrombus formation after flow via binding and aggregation of fluorescently-labelled platelets and fibrinogen. RESULTS AND CONCLUSIONS: The annexin V-thrombin conjugate induced rapid thrombosis (fibrin deposition) on irradiated endothelial cells under shear stress in the parallel-plate flow device. Unconjugated, non-targeting thrombin did not induce fibrin deposition. A synergistic interaction between radiation and conjugate dose was observed. Thrombosis was greatest at the highest combined doses of radiation (25 Gy) and conjugate (2.5 µg/mL). The parallel-plate flow system provides a rapid method to pre-test pro-thrombotic vascular targeting agents. These findings validate the translation of the annexin V-thrombin conjugate to pre-clinical studies.

Characteristics of CSF Velocity-Time Profile in Posttraumatic Syringomyelia.

BACKGROUND AND PURPOSE: The development of syringomyelia has been associated with changes in CSF flow dynamics in the spinal subarachnoid space. However, differences in CSF flow velocity between patients with posttraumatic syringomyelia and healthy participants remains unclear. The aim of this work was to define differences in CSF flow above and below a syrinx in participants with posttraumatic syringomyelia and compare the CSF flow with that in healthy controls. MATERIALS AND METHODS: Six participants with posttraumatic syringomyelia were recruited for this study. Phase-contrast MR imaging was used to measure CSF flow velocity at the base of the skull and above and below the syrinx. Velocity magnitudes and temporal features of the CSF velocity profile were compared with those in healthy controls. RESULTS: CSF flow velocity in the spinal subarachnoid space of participants with syringomyelia was similar at different locations despite differences in syrinx size and locations. Peak cranial and caudal velocities above and below the syrinx were not significantly different (peak cranial velocity, P = .9; peak caudal velocity, P = 1.0), but the peak velocities were significantly lower (P < .001, P = .007) in the participants with syringomyelia compared with matched controls. Most notably, the duration of caudal flow was significantly shorter (P = .003) in the participants with syringomyelia. CONCLUSIONS: CSF flow within the posttraumatic syringomyelia group was relatively uniform along the spinal canal, but there are differences in the timing of CSF flow compared with that in matched healthy controls. This finding supports the hypothesis that syrinx development may be associated with temporal changes in spinal CSF flow.

Inwardly rectifying potassium channel 4.1 expression in post-traumatic syringomyelia.

Post-traumatic syringomyelia (PTS) is a serious neurological disorder characterized by fluid filled cavities that develop in the spinal cord. PTS is thought to be caused by an imbalance between fluid inflow and outflow in the spinal cord, but the underlying mechanisms are unknown. The ion channel Kir4.1 plays an important role in the uptake of K(+) ions from the extracellular space and release of K(+) ions into the microvasculature, generating an osmotic gradient that drives water movement. Changes in Kir4.1 expression may contribute to disturbances in K(+) homeostasis and subsequently fluid imbalance. Here we investigated whether changes in Kir4.1 protein expression occur in PTS. Western blotting and immunohistochemistry were used to evaluate Kir4.1 and glial fibrillary acidic protein (GFAP) expression in a rodent model of PTS at 3 days, 1, 6 or 12 weeks post-surgery. In Western blotting experiments, Kir4.1 expression increased 1 week post-surgery at the level of the cavity. Immunohistochemical analysis examined changes in the spinal parenchyma directly in contact with the syrinx cavity. In these experiments, there was a significant decrease in Kir4.1 expression in PTS animals compared to controls at 3 days and 6 weeks post-surgery, while an up-regulation of GFAP in PTS animals was observed at 1 and 12 weeks. This suggests that while overall Kir4.1 expression is unchanged at these time-points, there are many astrocytes surrounding the syrinx cavity that are not expressing Kir4.1. The results demonstrate a disturbance in the removal of K(+) ions in tissue surrounding a post-traumatic syrinx cavity. It is possible this contributes to water accumulation in the injured spinal cord leading to syrinx formation or exacerbation of the underlying pathology.

Clinical Applications of Cine Balanced Steady-State Free Precession MRI for the Evaluation of the Subarachnoid Spaces.

The purpose of this article is to review the physiology of normal brain and spinal cord motion in the subarachnoid space, principles of cine balanced steady-state free precession (bSSFP) magnetic resonance imaging (MRI), clinical applications, and the pitfalls encountered with this technique. The brain and spinal cord are dynamic structures that move with each heartbeat due to transmitted arterial pulse waves. Conventional MRI sequences do not allow anatomic evaluation of the pulsatile movement of the neural structures in the subarachnoid space due to limitations in temporal resolution. Cine bSSFP MRI uses cardiac gating to evaluate dynamically the brain and spinal cord with high contrast and temporal resolution.Cine bSSFP can be used in the evaluation of idiopathic syringomyelia to assess an underlying treatable cause, including arachnoid bands, which are usually not well visualized with conventional MR sequences due to motion artifact. This MRI technique is also useful in the evaluation of intraspinal and intracranial arachnoid cysts and the degree of mass effect on the cord. Other applications include preoperative and postoperative evaluation of Chiari I malformation and the evaluation of lateral ventricular asymmetry. The major limitation of cine bSSFP is the presence of banding artifacts, which can be reduced by shimming and modifying other scan parameters.

Different responses of cavernous malformations and arteriovenous malformations to radiosurgery.

The vascular structure of cavernous malformations (CMs) and arteriovenous malformations (AVMs) is different and they have differing clinical responses to radiosurgery. The structural differences of irradiated and non-irradiated CMs and AVMs were examined to clarify their differential responses to radiosurgery. CMs showed a greater ratio of intraluminal diameter to vessel wall thickness and a lack of subendothelial fibroblasts, myofibroblasts and smooth muscle cells compared with AVMs. Partial proteinaceous clots (19-22% of lumen) formed in CM sinusoids after radiosurgery but complete vaso-occlusion did not occur for up to 6 years after radiosurgery. In contrast, complete vaso-occlusion (91-98% of lumen) by fibrin thrombi that are permanent clots was observed in AVM vessels. Radiation-induced neuronal loss, neurofibrillary degeneration of neurons and myelin fragmentation were typical in the surrounding brain tissue of the irradiated lesions. The different structure and cellular composition of CMs and AVMs is likely to influence their responses to radiosurgery.

Tensile radial stress in the spinal cord related to arachnoiditis or tethering: a numerical model.

Spinal arachnoiditis comprises fibrous scarring of the subarachnoid space, following spinal trauma or inflammation, and is often associated with syringomyelia. We hypothesised that cord-to-dura attachments could cause transient tensile cord radial stress, as pressure waves propagate. This was tested in a fluid-structure interaction model, simulating three types of cord tethering, with 'arachnoiditis' confined to a short mid-section of the cord. The annular system was excited abdominally with a short transient, and the resulting Young and Lamb waves and reflections were analysed. Radial mid-section tethering was less significant than axial tethering, which gave rise to tensile radial stress locally when the cord was not fixed cranially. Simulated as inextensible string connections to the dura, arachnoiditis caused both localised tensile radial stress and localised low pressure in the cord as the transient passed. The extent of these effects was sensitive to the relative stiffness of the dura and cord. Tensile radial stress may create a syrinx in previously normal cord tissue, and transiently lowered pressure may draw in interstitial fluid, causing the syrinx to enlarge if fluid exit is inhibited. The suggested mechanism could also explain the juxtaposition of syrinxes to regions of arachnoiditis.

CSF pathways: a review.

The treatment of patients with cerebrospinal fluid (CSF) pathway disorders (including hydrocephalus, syringomyelia, and arachnoid cysts), requires an understanding of CSF pathways and the cerebral water system. Although outcome with these disorders has improved, only limited treatment options remain available. Ongoing research has continued to improve our understanding of the cerebral water circulation and is starting to provide insight into the pathogenesis and potential treatment options of this common group of disorders. This article reviews current concepts of CSF function and pathways, following the journey of CSF from conception to absorption.

In vitro and in vivo tissue repair with laser-activated chitosan adhesive.

BACKGROUND AND OBJECTIVES: Sutures are currently the gold standard for wound closure but they are still unable to seal tissue and may induce scarring or inflammation. Biocompatible glues, based on polysaccharides such as chitosan, are a possible alternative to conventional wound closure. In this study, the adhesion of laser-activated chitosan films is investigated in vitro and in vivo. In particular we examine the effect of varying the laser power, as well as adding a natural cross-linker (genipin) to the adhesive composition. STUDY DESIGN/MATERIALS AND METHODS: Flexible and insoluble strips of chitosan films (surface area approximately 34 mm(2), thickness approximately 20 microm) were bonded to sheep intestine using several laser powers (0, 80, 120, and 160 mW) at 808-nm wavelength. The strength of repaired tissue was tested by a calibrated tensiometer to select the best power. A natural cross-linker (genipin) was also added to the film and the tissue repair strength compared with the strength of plain films. The adhesive was also bonded in vivo to the sciatic nerve of rats and the thermal damage induced by the laser assessed 4 days post-operatively. RESULTS: Chitosan adhesives successfully repaired intestine tissue, attaining a maximum repair strength of 14.7+/-4.3 kPa (n = 30) at the laser power of 120 mW. The chitosan-genipin films achieved lower repair strength (9.1+/-2.9 kPa). The laser caused partial demyelination of axons at the site of operation, but the myelinated axons retained a normal morphology proximally and distally. CONCLUSIONS: The chitosan adhesive effectively bonded to tissue causing only localized thermal damage in vivo, when the appropriate laser parameters were selected.

Focal spinal arachnoiditis increases subarachnoid space pressure: a computational study.

BACKGROUND: Enlarging fluid filled cystic cavitations form within the spinal cord in up to 28% of spinal cord injured patients. These post-traumatic syrinxes can cause neurological deterioration and current treatment results are unsatisfactory. Localized scar tissue (arachnoiditis) within the subarachnoid space at the level of injury has been suggested to be involved in the pathogenesis of syrinx formation. This study tests the hypothesis that pressure pulses in the subarachnoid space are accentuated adjacent to regions of arachnoiditis, which may drive fluid into the spinal cord and contribute to syrinx formation. METHODS: An axisymmetric, cylindrical computational fluid dynamics model was developed to represent the subarachnoid space under normal physiological conditions and in the presence of arachnoiditis. Cerebrospinal fluid flow into the model was estimated from magnetic resonance imaging flow studies. Arachnoiditis was modelled as a porous obstruction in the subarachnoid space. FINDINGS: Peak fluid pressures were higher above the obstruction than in the absence of obstruction. The peak pressures were strongly dependent on the permeability of the obstruction. INTERPRETATION: Elevations in subarachnoid space pressures due to arachnoiditis may facilitate fluid flow into the spinal cord, enhancing syrinx formation. This suggests that it may be worthwhile to investigate strategies that inhibit arachnoiditis or minimize systolic pressure peaks for treating or preventing syringomyelia.

The origins of syringomyelia: numerical models of fluid/structure interactions in the spinal cord.

A two-dimensional axi-symmetric numerical model is constructed of the spinal cord, consisting of elastic cord tissue surrounded by aqueous cerebrospinal fluid, in turn surrounded by elastic dura. The geometric and elastic parameters are simplified but of realistic order, compared with existing measurements. A distal reflecting site models scar tissue formed by earlier trauma to the cord, which is commonly associated with syrinx formation. Transients equivalent to both arterial pulsation and percussive coughing are used to excite wave propagation. Propagation is investigated in this model and one with a central canal down the middle of the cord tissue, and in further idealized versions of it, including a model with no cord, one with a rigid cord, one with a rigid dura, and a double-length untapered variant of the rigid-dura model. Analytical predictions for axial and radial wave-speeds in these different situations are compared with, and used to explain, the numerical outcomes. We find that the anatomic circumstances of the spinal cerebrospinal fluid cavity probably do not allow for significant wave steepening phenomena. The results indicate that wave propagation in the real cord is set by the elastic properties of both the cord tissue and the confining dura mater, fat, and bone. The central canal does not influence the wave propagation significantly.

Angiographic, hemodynamic and histological characterization of an arteriovenous fistula in rats.

BACKGROUND: Our understanding of the pathogenesis of arteriovenous malformations (AVMs) and arteriovenous fistulas (AVFs) has been limited by the lack of adequate animal models. In this study we evaluate the time course of angiographic, hemodynamic and histopathological changes in an arteriovenous fistula in rats as a potential model. METHODS: An arteriovenous fistula was created by a side-to-end anastomosis of the common carotid artery (CCA) to the external jugular vein (EJV). The animals underwent angiography of the fistula and were sacrificed 1, 7, 21, 42 or 90 days later. Flow and pressure measurements were performed in the CCA and ipsi- and contralateral EJV and detailed histological examination of whole mount sections of the fistula and cranium were done on fixed sections. Immunohistochemistry for CD31, smooth muscle alpha-actin and Ki-67 were performed. FINDINGS: Hemodynamic changes occur immediately after fistula formation creating a stable high flow, low resistant state. This induces a gradual increase in the inner diameter of the EJV and transverse sinus followed by a decrease in size of the transverse sinus. This decrease is associated with increased expression of alpha-actin in the wall of the sinus. The fistula becomes angiographically and histologically stable after 21 days. CONCLUSION: This model describes the time course of hemodynamic and histopathological changes after occur after AVF formation. Stabilization after 21 days makes it an attractive model for mechanistic and therapeutic studies of AVFs.

Syringomyelia and the arachnoid web.

Three cases of syringomyelia associated with arachnoid webs are reported. Each patient presented with progressive myelopathy and had thoracic syringes detected on magnetic resonance imaging (MRI). In one patient the web was also visible. At operation a thoracic arachnoid web was found, obstructing the subarachnoid compartment in each patient. One patient had intraoperative ultrasound, which demonstrated caudal web movement with each cardiac systole. The webs were divided and shunts inserted into the syringes. All patients improved clinically, and on follow-up MRI. Arachnoid webs are likely to represent a focal band of arachnoiditis and are difficult to visualise on standard preoperative MR imaging. A reduction in the subarachnoid space compliance with resultant increase in pulse pressure and potentiation of an arterial pulsation driven perivascular flow could explain the associated syringes. Treatment should be aimed at restoring compliance, and involve division of the web with or without shunt insertion.

Post-traumatic syringomyelia: a review.

More than a quarter of spinal cord injured patients develop syringes and many of these patients suffer progressive neurological deficits as a result of cyst enlargement. The mechanism of initial cyst formation and progressive enlargement are unknown, although arachnoiditis and persisting cord compression with disturbance of cerebrospinal fluid flow appear to be important aetiological factors. Current treatment options include correction of bony deformity, decompression of the spinal cord, division of adhesions, and shunting. Long-term improvement occurs in fewer than half of patients treated. Imaging evidence of a reduction in syrinx size following treatment does not guarantee symptomatic resolution or even prevention of further neurological loss. A better understanding of the causal mechanisms of syringomyelia is required to develop more effective therapy.

Excitotoxic model of post-traumatic syringomyelia in the rat.

STUDY DESIGN: A rat model was developed to elucidate the role of excitatory amino acids and spinal subarachnoid block in the genesis of post-traumatic syringomyelia. This excitotoxic model produces intramedullary cavities rather than the dilation of the central canal (canalicular syringomyelia) created by previous animal models. OBJECTIVES: To produce extracanalicular cysts in the rat spinal cord with quisqualic acid, a potent agonist of multiple excitatory amino acid receptors, and to compare the effects of excitotoxic injury only with that of excitotoxic injury and subarachnoid block with kaolin. SUMMARY OF BACKGROUND DATA: In post-traumatic syringomyelia, primary injury and excitotoxic cell death secondary to elevated levels of excitatory amino acids may initiate a pathologic process leading to the formation of spinal cavities. Subarachnoid block by arachnoiditis may promote enlargement of the cavities. METHODS: Three control rats received a unilateral injection of normal saline into the spinal cord, and another five rats received an injection of kaolin into the spinal subarachnoid space. Quisqualic acid was injected unilaterally into the spinal cord of 20 rats, and 13 additional rats received a unilateral injection of quisqualic acid into the spinal cord after injection of kaolin into the subarachnoid space. Histologic and immunocytochemical assessments were undertaken. RESULTS: In the control groups, no parenchymal cyst developed in any of the animals. Spinal cord cyst formation was observed in 16 of 19 animals in the quisqualic acid groups, but no cysts exceeding two segments in the length of the spinal cord developed in any of the rats. Much larger cavities were seen in 9 of 11 animals in the group with quisqualic acid and kaolin, and cysts exceeding two segments developed in all 9 of these (9/11; 82%). CONCLUSIONS: In post-traumatic syringomyelia, excitotoxic cell death occurring secondarily to elevated levels of excitatory amino acids may contribute to the pathologic process leading to the formation of spinal cord cysts. Subarachnoid block by arachnoiditis is likely to cause enlargement of the cavity.

Adenoviral gene transfer of nitric oxide synthase increases cerebral blood flow in rats.

OBJECTIVE: Depletion of nitric oxide may play a role in the development of vasospasm after aneurysmal subarachnoid hemorrhage. Replenishment of nitric oxide might be a useful treatment for vasospasm. Using rats, we performed intracisternal injections of replication-defective adenovirus containing the endothelial nitric oxide synthase (eNOS) gene and determined the localization of and effect on cerebral blood flow of transgene expression. METHODS: Rats underwent baseline measurement of cortical cerebral blood flow using laser Doppler flowmetry. Replication-defective adenovirus containing the Escherichia coli LacZ gene (Ad327beta-Gal, n = 2/time point) or the bovine eNOS gene (AdCD8-NOS, n = 4/time point) or physiological saline solution was injected into the cisterna magna. Cerebral blood flow was measured 1, 2, 4, 7, or 14 days later, and the animals were killed. Expression of beta-galactosidase activity from the LacZ gene was examined by histochemical staining and that of eNOS was examined by polymerase chain reaction assays of messenger ribonucleic acid. Brains were histopathologically examined for inflammation. RESULTS: Beta-galactosidase activity was observed throughout the leptomeninges and in some cells in the adventitia of small subarachnoid blood vessels in the Ad327beta-Gal-injected rats. Messenger ribonucleic acid for eNOS was detected in the leptomeninges and brainstem 1 and 2 days after injection of AdCD8-NOS. Rats injected with Ad327beta-Gal or physiological saline solution exhibited decreased cerebral blood flow beginning 2 days after virus injection and lasting up to 14 days after injection. Rats injected with AdCD8-NOS developed significant transient increases in cerebral blood flow 2 days after virus injection, followed by slight decreases in blood flow. There was inflammation in the subarachnoid space of all animals; the inflammation was qualitatively worse in animals injected with Ad327beta-Gal, compared with rats injected with AdCD8-NOS or saline solution. CONCLUSION: Intracisternal injection of replication-defective adenovirus containing the eNOS gene can transiently increase cerebral blood flow.

Subarachnoid hemorrhage as a cause of an adaptive response in cerebral arteries.

OBJECT: It is not known whether the factors responsible for vasospasm after subarachnoid hemorrhage (SAH) cause the cerebral arteries to be narrowed independent of the subarachnoid blood clot or whether the continued presence of clot is required for the entire time of vasospasm. The authors undertook the present study to investigate this issue. METHODS: To distinguish between these possibilities, bilateral SAH was induced in monkeys. The diameters of the monkeys' cerebral arteries were measured on angiograms obtained on Days 0 (the day of SAH), 1, 3, 5, 7, and 9. The subarachnoid blood clot was removed surgically on Day 1, 3, or 5 or, in control animals, was not removed until the animals were killed on Day 7 or 9. The concentrations of hemoglobins and adenosine triphosphate (ATP), substances believed to cause vasospasm, were measured in the removed clots and the contractile activity of the clots was measured in monkey basilar arteries in vitro. If the clot was removed 1 or 3 days after placement, vasospasm was significantly diminished 4 days after clot removal. Clot removal on Day 5 had no marked effect on vasospasm. There was a significant decrease over time in hemoglobin and ATP concentrations and in the contractile activity of the clots, although substantial hemoglobin and contractile activity was still present on Day 7. CONCLUSIONS: The authors infer from these results that vasospasm requires the presence of subarachnoid blood for at least 3 days, whereas by Day 5 vasospasm is less dependent on subarachnoid blood clot. Because the clot still contains substantial amounts of hemoglobin and contractile activity after 5 days, there may be an adaptive response in the cerebral arteries that allows them to relax in the presence of the stimulus that earlier caused contraction.