Babak S. Jahromi

Transmitter release increases intracellular calcium in perisynaptic schwann cells in situ

Glial cells isolated from the nervous system are sensitive to neurotransmitters and may therefore be involved in synaptic transmission. The sensitivity of individual perisynaptic Schwann cells to activity of a single synapse was investigated, in situ, at the frog neuromuscular junction by monitoring changes in intracellular Ca2+ in the Schwann cells. Motor nerve stimulation induced an increase in intracellular Ca2+ in these Schwann cells; this increase was greatly reduced when transmitter release was blocked. Furthermore, local application of the cotransmitters acetylcholine and ATP evoked Ca2+ responses even in the absence of extracellular Ca2+. Successive trains of nerve stimuli or applications of transmitters resulted in progressively smaller Ca2+ responses. We conclude that transmitter released during synaptic activity can evoke release of intracellular Ca2+ in perisynaptic Schwann cells. This Ca2+ signal may play a role in the maintenance or modulation of a synapse. These data show that synaptic transmission involves three cellular components with both postsynaptic and glial components responding to transmitter secretion.

CLINICAL AND ANGIOGRAPHIC OUTCOME AFTER ENDOVASCULAR MANAGEMENT OF GIANT INTRACRANIAL ANEURYSMS

OBJECTIVEGiant (≥25 mm) intracranial aneurysms (IA) have an extremely poor natural history and continue to confound modern techniques for management. Currently, there is a dearth of large series examining endovascular treatment of giant IAs only. METHODSWe reviewed long-term clinical and radiological outcome from a series of 39 consecutive giant IAs treated with endovascular repair in 38 patients at 2 tertiary referral centers. Data were evaluated in 3 ways: on a per-treatment session basis for each aneurysm, at 30 days after each patients final treatment, and at the last known follow-up examination. RESULTSTen (26%) aneurysms were ruptured. At the last angiographic follow-up examination (21.5 ± 22.9 months), 95% or higher and 100% occlusion rates were documented in 64 and 36% of aneurysms, respectively, with parent vessel preservation maintained in 74%. Stents were required in 25 aneurysms. Twenty percent of treatment sessions resulted in permanent morbidity, and death within 30 days occurred after 8% of treatment sessions. On average, 1.9 ± 1.1 sessions were required to treat each aneurysm, with a resulting cumulative per-patient mortality of 16% and morbidity of 32%. At the last known clinical follow-up examination (mean, 24.8 ± 24.8 months), 24 (63%) patients had Glasgow Outcome Scale scores of 4 or 5 (“good” or “excellent”), 10 patients had worsened neurological function from baseline (26% morbidity), and 11 had died (29% mortality). CONCLUSIONWe present what is to our knowledge the largest series to date evaluating outcome after consecutive giant IAs treated with endovascular repair. Giant IAs carry a high risk for surgical or endovascular intervention. We hope critical and honest evaluation of treatment results will ensure continued improvement in patient care.

Novel mechanism of endothelin-1-induced vasospasm after subarachnoid hemorrhage

Cerebral vasospasm is a major cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). It is a sustained constriction of the cerebral arteries that can be reduced by endothelin (ET) receptor antagonists. Voltage-gated Ca2+ channel antagonists such as nimodipine are relatively less effective. Endothelin-1 is not increased enough after SAH to directly cause the constriction, so we sought alternate mechanisms by which ET-1 might mediate vasospasm. Vasospasm was created in dogs, and the smooth muscle cells were studied molecularly, electro-physiologically, and by isometric tension. During vasospasm, ET-1, 10 nmol/L, induced a nonselective cation current carried by Ca2+ in 64% of cells compared with in only 7% of control cells. Nimodipine and 2-aminoethoxydiphenylborate (a specific antagonist of store-operated channels) had no effect, whereas SKF96365 (a nonspecific antagonist of nonselective cation channels) decreased this current in vasospastic smooth muscle cells. Transient receptor potential (TRP) proteins may mediate entry of Ca2+ through nonselective cationic pathways. We tested their role by incubating smooth muscle cells with anti-TRPC1 or TRPC4, both of which blocked ET-1-induced currents in SAH cells. Anti-TRPC5 had no effect. Anti-TRPC1 also inhibited ET-1 contraction of SAH arteries in vitro. Quantitative polymerase chain reaction and Western blotting of seven TRPC isoforms found increased expression of TRPC4 and a novel splice variant of TRPC1 and increased protein expression of TRPC4 and TRPC1. Taken together, the results support a novel mechanism whereby ET-1 significantly increases Ca2+ influx mediated by TRPC1 and TRPC4 or their heteromers in smooth muscle cells, which promotes development of vasospasm after SAH.

Muscarinic Ca2+ responses resistant to muscarinic antagonists at perisynaptic schwann cells of the frog neuromuscular junction

1 Acetylcholine causes a rise of intracellular Ca2+ in perisynaptic Schwann cells (PSCs) of the frog neuromuscular junction. The signalling pathway was characterized using the fluorescent Ca2+ indicator fluo‐3 and fluorescence microscopy. 2 Nicotinic antagonists had no effect on Ca2+responses evoked by ACh and no Ca2+ responses were evoked with the nicotinic agonist nicotine. The muscarinic agonists muscarine and oxotremorine‐M induced Ca2+ signals in PSCs. 3 Ca2+ responses remained unchanged when extracellular Ca2+ was removed, indicating that they are due to the release of Ca2+ from internal stores. Incubation with pertussis toxin did not alter the Ca2+ signals induced by muscarine, but did block depression of transmitter release induced by adenosine and prevented Ca2+ responses in PSCs induced by adenosine. 4 The general muscarinic antagonists atropine, quinuclidinyl benzilate and N‐methyl‐scopolamine failed to block Ca2+ responses to muscarinic agonists. Atropine (at 20000‐fold excess concentration) also failed to reduce the proportion of cells responding to a threshold muscarine concentration sufficient to cause responses in less than 50% of cells. Only the allosteric, non‐specific blocker, gallamine (1–10 μm) was effective in blocking muscarine‐induced Ca2+ responses. 5 In preparations denervated 7 days prior to experiments, low concentrations of atropine reversibly and completely blocked Ca2+ responses to muscarine. 6 The lack of blockade by general muscarinic antagonists in innervated, in situ preparations suggests that muscarinic Ca2+ responses at PSCs are not mediated by any of the five known muscarinic receptors or that post‐translational modification prevented antagonist binding.

Voltage-dependent calcium channels of dog basilar artery

Electrophysiological and molecular characteristics of voltage‐dependent calcium (Ca2+) channels were studied using whole‐cell patch clamp, polymerase chain reaction and Western blotting in smooth muscle cells freshly isolated from dog basilar artery. Inward currents evoked by depolarizing steps from a holding potential of –50 or –90 mV in 10 mm barium consisted of low‐ (LVA) and high‐voltage activated (HVA) components. LVA current comprised more than half of total current in 24 (12%) of 203 cells and less than 10% of total current in 52 (26%) cells. The remaining cells (127 cells, 62%) had LVA currents between one tenth and one half of total current. LVA current was rapidly inactivating, slowly deactivating, inhibited by high doses of nimodipine and mibefradil (> 0.3 μm), not affected by ω‐agatoxin GVIA (γ100 nm), ω‐conotoxin IVA (1 μm) or SNX‐482 (200 nm) and probably carried by T‐type Ca2+ channels based on the presence of messenger ribonucleic acid (mRNA) and protein for Cav3.1 and Cav3.3α1 subunits of these channels. LVA currents exhibited window current with a maximum of 13% of the LVA current at –37.4 mV. HVA current was slowly inactivating and rapidly deactivating. It was inhibited by nimodipine (IC50= 0.018 μm), mibefradil (IC50= 0.39 μm) and ω‐conotoxin IV (1 μm). Smooth muscle cells also contained mRNA and protein for L‐ (Cav1.2 and Cav1.3), N‐ (Cav2.2) and T‐type (Cav3.1 and Cav3.3) α1 Ca2+ channel subunits. Confocal microscopy showed Cav1.2 and Cav1.3 (L‐type), Cav2.2 (N‐type) and Cav3.1 and Cav3.3 (T‐type) protein in smooth muscle cells. Relaxation of intact arteries under isometric tension in vitro to nimodipine (1 μm) and mibefradil (1 μm) but not to ω‐agatoxin GVIA (100 nm), ω‐conotoxin IVA (1 μm) or SNX‐482 (1 μm) confirmed the functional significance of L‐ and T‐type voltage‐dependent Ca2+ channel subtypes but not N‐type. These results show that dog basilar artery smooth muscle cells express functional voltage‐dependent Ca2+ channels of multiple types.

Molecular Profile of Vascular Ion Channels After Experimental Subarachnoid Hemorrhage

Cerebral vasospasm is a transient, delayed constriction of cerebral arteries that occurs after subarachnoid hemorrhage (SAH). Smooth muscle cells show impaired relaxation after SAH, which may be caused by a defect in the ionic mechanisms regulating smooth muscle membrane potential and Ca2+ permeability. We tested this hypothesis by examining changes in expression of mRNA and protein for ion channels in the basilar arteries of dogs after SAH using quantitative real-time polymerase chain reaction (PCR) and western blotting. SAH was associated with a significant reduction in basilar artery diameter to 41 ± 8% of pre-SAH diameter (P < 0.001) after 7 days. There was significant downregulation of the voltage-gated K+ channel Kv 2.2 (65% reduction in mRNA, P < 0.001; 49% reduction in protein, P < 0.05) and the β1 subunit of the large-conductance, Ca2+-activated K+ (BK) channel (53% reduction in mRNA, P < 0.02). There was no change in BK β1 subunit protein. Changes in mRNA levels of Kv 2.2 and the BK-β1 subunit correlated with the degree of vasospasm (r2 = 0.490 and 0.529 respectively, P < 0.05). The inwardly rectifying K+ (Kir) channel Kir 2.1 was upregulated (234% increase in mRNA, P < 0.001; 350% increase in protein, P < 0.001). There was no significant change in mRNA expression of L- type Ca2+ channels and the BK-α subunit. These data suggest that K+ channel dysfunction may contribute to the pathogenesis of cerebral vasospasm.

Clinical factors associated with venous thromboembolism risk in patients undergoing craniotomy

OBJECT Patients undergoing craniotomy are at risk for developing venous thromboembolism (VTE). The safety of anticoagulation in these patients is not clear. The authors sought to identify risk factors predictive of VTE in patients undergoing craniotomy. METHODS The authors reviewed a national surgical quality database, the American College of Surgeons National Surgical Quality Improvement Program. Craniotomy patients were identified by current procedural terminology code. Clinical factors were analyzed to identify associations with VTE. RESULTS Four thousand eight hundred forty-four adult patients who underwent craniotomy were identified. The rate of VTE in the cohort was 3.5%, including pulmonary embolism in 1.4% and deep venous thrombosis in 2.6%. A number of factors were found to be statistically significant in multivariate binary logistic regression analysis, including craniotomy for tumor, transfer from acute care hospital, age ≥ 60 years, dependent functional status, tumor involving the CNS, sepsis, emergency surgery, surgery time ≥ 4 hours, postoperative urinary tract infection, postoperative pneumonia, on ventilator ≥ 48 hours postoperatively, and return to the operating room. Patients were assigned a score based on how many of these factors they had (minimum score 0, maximum score 12). Increasing score was predictive of increased VTE incidence, as well as risk of mortality, and time from surgery to discharge. CONCLUSIONS Patients undergoing craniotomy are at low risk of developing VTE, but this risk is increased by preoperative medical comorbidities and postoperative complications. The presence of more of these clinical factors is associated with progressively increased VTE risk; patients possessing a VTE Risk Score of ≥ 5 had a greater than 20-fold increased risk of VTE compared with patients with a VTE score of 0.

Voltage-gated K + channel dysfunction in myocytes from a dog model of subarachnoid hemorrhage

Delayed cerebral vasospasm after subarachnoid hemorrhage is primarily due to sustained contraction of arterial smooth muscle cells. Its pathogenesis remains unclear. The degree of arterial constriction is regulated by membrane potential that in turn is determined predominately by K+ conductance (GK). Here, we identified the main voltage-gated K+ (Kv) channels contributing to outward delayed rectifier currents in dog basilar artery smooth muscle as Kv2 class through a combination of electrophysiological and pharmacological methods. Kv2 current density was nearly halved in vasospastic myocytes after subarachnoid hemorrhage (SAH) in dogs, and Kv2.1 and Kv2.2 were downregulated in vasospastic myocytes when examined by quantitative mRNA, Western blotting, and immunohistochemistry. Vasospastic myocytes were depolarized and had a smaller contribution of GK toward maintenance of their membrane potential. Pharmacological block of Kv current in control myocytes mimicked the depolarization observed in vasospastic arteries. The degree of membrane depolarization was found to be compatible with the amount of vasoconstriction observed after SAH. We conclude that Kv2 dysfunction after SAH contributes to the pathogenesis of delayed cerebral vasospasm. This may confer a novel target for treatment of delayed cerebral vasospasm.

Expression and function of inwardly rectifying potassium channels after experimental subarachnoid hemorrhage

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is because of smooth muscle contraction, although the mechanism of this contraction remains unresolved. Membrane potential controls the contractile state of arterial myocytes by gating voltage-sensitive calcium channels and is in turn primarily controlled by K+ ion conductance through several classes of K+ channels. We characterized the role of inwardly rectifying K+ (KIR) channels in vasospasm. Vasospasm was created in dogs using the double-hemorrhage model of SAH. Electrophysiological, real-time quantitative reverse-transcriptase polymerase chain reaction, Western blotting, immunohistochemistry, and isometric tension techniques were used to characterize the expression and function of KIR channels in normal and vasospastic basilar artery 7 days after SAH. Subarachnoid hemorrhage resulted in severe vasospasm of the basilar artery (mean of 61% ± 5% reduction in diameter). Membrane potential of pressurized vasospastic basilar arteries was significantly depolarized compared with control arteries (+-46 ± 1.4 mV versus −29.8 ± 1.8 mV, respectively, P < 0.01). In whole-cell patch clamp of enzymatically isolated basilar artery myocytes, average KIR conductance was 1.6 ± 0.5 pS/pF in control cells and 9.2 ± 2.2 pS/pF in SAH cells (P = 0.007). Blocking KiR channels with BaCI2 (0.1 mmol/L) resulted in significantly greater membrane depolarization in vasospastic compared with normal myocytes. Expression of KIR 2.1 messenger ribonucleic acid (mRNA) was increased after SAH. Western blotting and immunohistochemistry also showed increased expression of KIR protein in vasospastic smooth muscle. Blockage of KIR channels in arteries under isometric tension produced a greater contraction in SAH than in control arteries. These results document increased expression of KIR 2.1 mRNA and protein during vasospasm after experimental SAH and suggest that this increase is a functionally significant adaptive response acting to reduce vasospasm.

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

SummaryBackground. 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 α-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 α-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.