Quantifying Intracranial Aneurysm Wall Permeability for Risk Assessment Using Dynamic Contrast-Enhanced MRI: A Pilot Study.

BACKGROUND AND PURPOSE: Pathological changes in the intracranial aneurysm wall may lead to increases in its permeability; however the clinical significance of such changes has not been explored. The purpose of this pilot study was to quantify intracranial aneurysm wall permeability (K(trans), VL) to contrast agent as a measure of aneurysm rupture risk and compare these parameters against other established measures of rupture risk. We hypothesized K(trans) would be associated with intracranial aneurysm rupture risk as defined by various anatomic, imaging, and clinical risk factors. MATERIALS AND METHODS: Twenty-seven unruptured intracranial aneurysms in 23 patients were imaged with dynamic contrast-enhanced MR imaging, and wall permeability parameters (K(trans), VL) were measured in regions adjacent to the aneurysm wall and along the paired control MCA by 2 blinded observers. K(trans) and VL were evaluated as markers of rupture risk by comparing them against established clinical (symptomatic lesions) and anatomic (size, location, morphology, multiplicity) risk metrics. RESULTS: Interobserver agreement was strong as shown in regression analysis (R(2) > 0.84) and intraclass correlation (intraclass correlation coefficient >0.92), indicating that the K(trans) can be reliably assessed clinically. All intracranial aneurysms had a pronounced increase in wall permeability compared with the paired healthy MCA (P < .001). Regression analysis demonstrated a significant trend toward an increased K(trans) with increasing aneurysm size (P < .001). Logistic regression showed that K(trans) also predicted risk in anatomic (P = .02) and combined anatomic/clinical (P = .03) groups independent of size. CONCLUSIONS: We report the first evidence of dynamic contrast-enhanced MR imaging-modeled contrast permeability in intracranial aneurysms. We found that contrast agent permeability across the aneurysm wall correlated significantly with both aneurysm size and size-independent anatomic risk factors. In addition, K(trans) was a significant and size-independent predictor of morphologically and clinically defined high-risk aneurysms.

Plasmalemmal repair of severed neurites of PC12 cells requires Ca(2+) and synaptotagmin.

Ca(2+) and synaptotagmin (a Ca(2+)-binding protein that regulates axolemmal fusion of synaptic vesicles) play essential roles in the repair of axolemmal damage in invertebrate giant axons. We now report that neurites of a rat pheochromocytoma (PC12) cell line transected and maintained in a serum medium form a dye barrier (exclude an external hydrophilic fluorescent dye) and survive for 24-hr posttransection (based on morphology and retention of another hydrophilic dye internally loaded at 6-hr posttransection). Some (25%) transected neurites that form a dye barrier regrow. Most (83%) neurites transected in a saline solution containing divalent cations (PBS(++)) also exclude entry of an externally placed hydrophilic fluorescent dye at 15-min posttransection. In contrast, only 14 or 17% of neurites maintained in a divalent cation-free solution (PBS(=)) or in PBS(=) + Mg(2+), respectively, form a dye barrier. Neurites that do not form a dye barrier do not survive for 24 hr. When PC12 neurites are loaded with an antibody to squid synaptotagmin, most (81%) antibody-loaded neurites do not form a dye barrier, whereas most (>/=81%) neurites loaded with heat-inactivated antibody or preimmune IgG do form a barrier. These data show that: 1) transected neurites of PC12 cells have mechanism(s) for plasmalemmal repair (dye barrier formation and survival); 2) Ca(2+) is necessary for dye barrier formation, which occurs minutes after transection and is necessary for survival and regrowth; and 3) synaptotagmin is an essential mediator of barrier formation. The similarity in the requirements for plasmalemmal repair in this mammalian cell preparation with those reported previously for invertebrate axons suggests that mechanisms necessary for plasmalemmal repair have been conserved phylogenetically.

Barrier permeability at cut axonal ends progressively decreases until an ionic seal is formed.

After axonal severance, a barrier forms at the cut ends to rapidly restrict bulk inflow and outflow. In severed crayfish axons we used the exclusion of hydrophilic, fluorescent dye molecules of different sizes (0.6-70 kDa) and the temporal decline of ionic injury current to levels in intact axons to determine the time course (0-120 min posttransection) of barrier formation and the posttransection time at which an axolemmal ionic seal had formed, as confirmed by the recovery of resting and action potentials. Confocal images showed that the posttransection time of dye exclusion was inversely related to dye molecular size. A barrier to the smallest dye molecule formed more rapidly (<60 min) than did the barrier to ionic entry (>60 min). These data show that axolemmal sealing lacks abrupt, large changes in barrier permeability that would be expected if a seal were to form suddenly, as previously assumed. Rather, these data suggest that a barrier forms gradually and slowly by restricting the movement of molecules of progressively smaller size amid injury-induced vesicles that accumulate, interact, and form junctional complexes with each other and the axolemma at the cut end. This process eventually culminates in an axolemmal ionic seal, and is not complete until ionic injury current returns to baseline levels measured in an undamaged axon.

Axolemmal repair requires proteins that mediate synaptic vesicle fusion.

A damaged cell membrane is repaired by a seal that restricts entry or exit of molecules and ions to that of the level passing through an undamaged membrane. Seal formation requires elevation of intracellular Ca(2+) and, very likely, protein-mediated fusion of membranes. Ca(2+) also regulates the interaction between synaptotagmin (Syt) and syntaxin (Syx), which is thought to mediate fusion of synaptic vesicles with the axolemma, allowing transmitter release at synapses. To determine whether synaptic proteins have a role in sealing axolemmal damage, we injected squid and crayfish giant axons with an antibody that inhibits squid Syt from binding Ca(2+), or with another antibody that inhibits the Ca(2+)-dependent interaction of squid Syx with the Ca(2+)-binding domain of Syt. Axons injected with antibody to Syt did not seal, as assessed at axonal cut ends by the exclusion of extracellular hydrophilic fluorescent dye using confocal microscopy, and by the decay of extracellular injury current compared to levels measured in uninjured axons using a vibrating probe technique. In contrast, axons injected with either denatured antibody to Syt or preimmune IgG did seal. Similarly, axons injected with antibody to Syx did not seal, but did seal when injected with either denatured antibody to Syx or preimmune IgG. These results indicate an essential involvement of Syt and Syx in the repair (sealing) of severed axons. We suggest that vesicles, which accumulate and interact at the injury site, re-establish axolemmal continuity by Ca(2+)-induced fusions mediated by proteins such as those involved in neurotransmitter release.

Anomalies associated with dye exclusion as a measure of axolemmal repair in invertebrate axons.

After axonal injury, dye exclusion is often used as a measure of the re-establishment of a structural barrier. We now report that this use of dye exclusion is equivocal in two situations. (1) When a negatively-charged hydrophilic fluorescent dye (HFD) was placed in the physiological saline (PS) surrounding a crayfish medial giant axon (CMGA) before transection, this dye did not readily diffuse into the cut ends after transection whereas uncharged or neutralized dyes did do so. (2) When axoplasm flowed out of the cut ends of a transected squid giant axon (SGA), this outflow markedly slowed hydrophilic fluorescent dyes from diffusing into the cut ends. These anomalies suggest that dye exclusion by an injured axon does not always indicate that a structural barrier has formed. Therefore, dye assessments of axonal repair require control experiments that rule out anomalous exclusion due to dye interactions (biochemical and fluid dynamics) with components (axoplasm, axolemma, glial sheath, etc.) of the particular axon under study.

Endocytotic formation of vesicles and other membranous structures induced by Ca2+ and axolemmal injury.

Vesicles and/or other membranous structures that form after axolemmal damage have recently been shown to repair (seal) the axolemma of various nerve axons. To determine the origin of such membranous structures, (1) we internally dialyzed isolated intact squid giant axons (GAs) and showed that elevation of intracellular Ca2+ >100 microM produced membranous structures similar to those in axons transected in Ca2+-containing physiological saline; (2) we exposed GA axoplasm to Ca2+-containing salines and observed that membranous structures did not form after removing the axolemma and glial sheath but did form in severed GAs after >99% of their axoplasm was removed by internal perfusion; (3) we examined transected GAs and crayfish medial giant axons (MGAs) with time-lapse confocal fluorescence microscopy and showed that many injury-induced vesicles formed by endocytosis of the axolemma; (4) we examined the cut ends of GAs and MGAs with electron microscopy and showed that most membranous structures were single-walled at short (5-15 min) post-transection times, whereas more were double- and multi-walled and of probable glial origin after longer (30-150 min) post-transection times; and (5) we examined differential interference contrast and confocal images and showed that large and small lesions evoked similar injury responses in which barriers to dye diffusion formed amid an accumulation of vesicles and other membranous structures. These and other data suggest that Ca2+ inflow at large or small axolemmal lesions induces various membranous structures (including endocytotic vesicles) of glial or axonal origin to form, accumulate, and interact with each other, preformed vesicles, and/or the axolemma to repair the axolemmal damage.

Calpain activity promotes the sealing of severed giant axons.

A barrier (seal) must form at the cut ends of a severed axon if a neuron is to survive and eventually regenerate. Following severance of crayfish medial giant axons in physiological saline, vesicles accumulate at the cut end and form a barrier (seal) to ion and dye diffusion. In contrast, squid giant axons do not seal, even though injury-induced vesicles form after axonal transection and accumulate at cut axonal ends. Neither axon seals in Ca2+-free salines. The addition of calpain to the bath saline induces the sealing of squid giant axons, whereas the addition of inhibitors of calpain activity inhibits the sealing of crayfish medial giant axons. These complementary effects involving calpain in two different axons suggest that endogenous calpain activity promotes plasmalemmal repair by vesicles or other membranes which form a plug or a continuous membrane barrier to seal cut axonal ends.

Repair of plasmalemmal lesions by vesicles.

Crayfish medial giant axons (MGAs) transected in physiological saline form vesicles which interact with each other, pre-existing vesicles, and/or with the plasmalemma to form an electrical and a physical barrier that seals a cut axonal end within 60 min. The formation of this barrier (seal) was assessed by measuring the decay of injury current at the cut end; its location at the cut end was determined by the exclusion of fluorescent hydrophilic dye at the cut end. When a membrane-incorporating styryl dye was placed in the bath prior to axonal transection and a hydrophilic dye was placed in the bath just after axonal transection, many vesicles near the barrier at the cut axonal end had their limiting membrane labeled with the styryl dye and their contents labeled with the hydrophilic dye, indicating that these vesicles originated from the axolemma by endocytosis. This barrier does not form in Ca2+-free salines. Similar collections of vesicles have been observed at regions of plasmalemmal damage in many cell types. From these and other data, we propose that plasmalemmal lesions in most eukaryotic cells (including axons) are repaired by vesicles, at least some of which arise by endocytosis induced by Ca2+ inflow resulting from the plasmalemmal damage. We describe several models by which vesicles could interact with each other and/or with intact or damaged regions of the plasmalemma to repair small (1-30 microm) plasmalemmal holes or a complete transection of the plasmalemma.