AAV9-Stathmin1 gene delivery improves disease phenotype in an intermediate mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a devastating infantile genetic disorder caused by the loss of survival motor neuron (SMN) protein that leads to premature death due to loss of motor neurons and muscle atrophy. The approval of an antisense oligonucleotide therapy for SMA was an important milestone in SMA research; however, effective next-generation therapeutics will likely require combinatorial SMN-dependent therapeutics and SMN-independent disease modifiers. A recent cross-disease transcriptomic analysis identified Stathmin-1 (STMN1), a tubulin-depolymerizing protein, as a potential disease modifier across different motor neuron diseases, including SMA. Here, we investigated whether viral-based delivery of STMN1 decreased disease severity in a well-characterized SMA mouse model. Intracerebroventricular delivery of scAAV9-STMN1 in SMA mice at P2 significantly increased survival and weight gain compared to untreated SMA mice without elevating Smn levels. scAAV9-STMN1 improved important hallmarks of disease, including motor function, NMJ pathology and motor neuron cell preservation. Furthermore, scAAV9-STMN1 treatment restored microtubule networks and tubulin expression without affecting tubulin stability. Our results show that scAAV9-STMN1 treatment improves SMA pathology possibly by increasing microtubule turnover leading to restored levels of stable microtubules. Overall, these data demonstrate that STMN1 can significantly reduce the SMA phenotype independent of restoring SMN protein and highlight the importance of developing SMN-independent therapeutics for the treatment of SMA.

CRISPR/Cas9 mediated generation of an ovine model for infantile neuronal ceroid lipofuscinosis (CLN1 disease).

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating monogenetic lysosomal disorders that affect children and young adults with no cure or effective treatment currently available. One of the more severe infantile forms of the disease (INCL or CLN1 disease) is due to mutations in the palmitoyl-protein thioesterase 1 (PPT1) gene and severely reduces the child's lifespan to approximately 9 years of age. In order to better translate the human condition than is possible in mice, we sought to produce a large animal model employing CRISPR/Cas9 gene editing technology. Three PPT1 homozygote sheep were generated by insertion of a disease-causing PPT1 (R151X) human mutation into the orthologous sheep locus. This resulted in a morphological, anatomical and biochemical disease phenotype that closely resembles the human condition. The homozygous sheep were found to have significantly reduced PPT1 enzyme activity and accumulate autofluorescent storage material, as is observed in CLN1 patients. Clinical signs included pronounced behavioral deficits as well as motor deficits and complete loss of vision, with a reduced lifespan of 17 ± 1 months at a humanely defined terminal endpoint. Magnetic resonance imaging (MRI) confirmed a significant decrease in motor cortical volume as well as increased ventricular volume corresponding with observed brain atrophy and a profound reduction in brain mass of 30% at necropsy, similar to alterations observed in human patients. In summary, we have generated the first CRISPR/Cas9 gene edited NCL model. This novel sheep model of CLN1 disease develops biochemical, gross morphological and in vivo brain alterations confirming the efficacy of the targeted modification and potential relevance to the human condition.

Intraperitoneal delivery of a novel drug-like compound improves disease severity in severe and intermediate mouse models of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder that causes progressive muscle weakness and is the leading genetic cause of infant mortality worldwide. SMA is caused by the loss of survival motor neuron 1 (SMN1). In humans, a nearly identical copy gene is present, called SMN2. Although SMN2 maintains the same coding sequence, this gene cannot compensate for the loss of SMN1 because of a single silent nucleotide difference in SMN2 exon 7. SMN2 primarily produces an alternatively spliced isoform lacking exon 7, which is critical for protein function. SMN2 is an important disease modifier that makes for an excellent target for therapeutic intervention because all SMA patients retain SMN2. Therefore, compounds and small molecules that can increase SMN2 exon 7 inclusion, transcription and SMN protein stability have great potential for SMA therapeutics. Previously, we performed a high throughput screen and established a class of compounds that increase SMN protein in various cellular contexts. In this study, a novel compound was identified that increased SMN protein levels in vivo and ameliorated the disease phenotype in severe and intermediate mouse models of SMA.

Risk factors of atherosclerosis and saphenous vein endothelial function.

BACKGROUND: Impaired vasomotor function has been suggested as playing a role in the pathophysiology of atherosclerosis and it may also affect the late patency of bypass grafts. We evaluated, in vitro, the influence of risk factors of atherosclerosis on saphenous vein endothelial function in patients with cardiovascular diseases. METHODS: Forty-five saphenous vein rings with intact (E+) and denuded endothelium (E-) were studied. The following drugs were used: norepinephrine (NE), acetylcholine (Ach), histamine (H) and serotonine (5-HT). RESULTS: Contraction to norepinephrine (n=15) showed a maximal tension of 783+/-115 percent that was increased in diabetics, smokers, and patients with hypertension. There was a wide range of response to acetylcholine in rings with intact endothelium (n=25), (mean relaxation 16.4+/-1.7 percent, ranging from -22.2 percent to 45 percent) with relaxation (26+/-1.1 percent) and contraction (-11+/-1.2 percent); relaxation was reduced in patients with hypertension and in diabetics (7.4+/-2.6 percent vs non diabetics 24.4+/-1.73 percent; p<0.01). Five of the 12 veins from diabetics exibited contraction (10+/-1.48 percent). Histamine (n=15) caused moderate relaxation at low doses (25+/-2.46 percent) followed by contraction at higher concentrations (184+/-5.7 percent). This was greater in diabetics (193+/-6.8 percent vs non diabetics 157+/-5.3 percent; p=0.045) while in preparations without endothelium (n=10) only relaxation was obtained (45+/-2.89 percent). Contraction (242+/-7.4 percent) was observed in response to serotonine (n=15) that was not affected by endothelial removal. In this study saphenous vein: (1) exhibited a wide range of responses to acetylcholine; (2) evoked marked contraction to norepinephrine and serotonine; (3) elicited contraction in response to histamine that was endothelium-dependent, suggesting the production or the release of an endothelium-derived-contracting-factor (EDCF). CONCLUSIONS: Saphenous vein is able to secrete a contracting factor in patients with risk factors of atherosclerosis and above all diabetes. The mechanisms that regulate the balance between the relaxing and contracting factors and how the endothelial cells become the source of the substances with vasoconstrictor activity remain to be determined.

Surgical reconstruction of the extracranial vertebral artery: management and outcome.

PURPOSE: The purpose of this study was to identify the risk and outcome of reconstruction of the extracranial vertebral artery (ECVA). METHOD: The study was conducted as a retrospective review of 369 consecutive ECVA reconstructions. RESULTS: The clinical presentations consisted of hemispheric symptoms alone in 4% of the cases, hemispheric and vertebrobasilar symptoms in 30%, and vertebrobasilar symptoms alone in 60%. The cause of the lesion was atherosclerosis (n = 300), extrinsic compression (n = 42), dissection (n = 7), radiation arteritis (n = 5), intimal hyperplasia (n = 3), fibromuscular dysplasia (n = 2), previous surgical ligation (n = 3), aneurysm (n = 2), and other (n = 5). All the patients underwent preoperative arteriography. There were 252 proximal ECVA reconstructions (218 transpositions, 42 bypass grafting procedures, and two other) and 117 distal ECVA reconstructions (85 bypass grafting procedures, 25 transpositions, and seven other). In 83 patients, the ECVA operation was performed concomitant with a carotid or supraaortic trunk reconstruction. This series was analyzed in two separate sets: before 1991 (n = 215), when changes in indications and management were occurring; and after 1991 (n = 154), when we acquired a dedicated anesthesia team and digital arteriography in the operating room and established uniform protocols for the management of ECVA disease. The stroke, death, and stroke/death rates for the period before 1991 were, respectively, 4. 1%, 3.2% and 5.1%. The stroke, death, and stroke/death rates for the period after 1991 were, respectively, 1.9%, 0.6% and 1.9%. The patency rate at 5 years was 80%. The survival rate at 5 years was 70%. Most of the deaths during the follow-up period were caused by cardiac disease. Among the survivors, the protection rate from stroke was 97%. CONCLUSION: The changes in operative selection and management have improved the results of ECVA reconstruction. The data reported for ECVA reconstruction in patients who underwent operation since 1991 reflect the outcome of ECVA reconstruction today. In our experience, a reconstruction of the ECVA is less risky than a carotid reconstruction.

Aortic thrombosis associated with cocaine use: report of two cases.

Cocaine use has been associated with many vascular complications which may involve the carotid, coronary, and renal vascular beds. Cocaine may also cause venous thrombosis. This report describes a new entity of cocaine-induced aortic thrombosis. On the basis of clinical findings and response to treatment, a therapeutic algorithm is presented.

Cervical reconstruction of the supra-aortic trunks: a 16-year experience.

PURPOSE: The purpose of this study was to review 182 consecutive cervical reconstructions of supra-aortic trunks, which were performed over a 16-year period. METHODS: A total of 182 innominate, common carotid, or subclavian arteries were reconstructed with a cervical approach in 173 patients aged 23 days to 83 years. Indications included hemispheric (n = 79), vertebrobasilar (n = 56), upper extremity (24), and internal mammary/cardiac ischemia (n = 5), asymptomatic severe common carotid disease (n = 33), or other (n = 3). Primary atherosclerotic innominate (n = 6), common carotid (n = 84), and subclavian (n = 66) lesions underwent reconstruction. Thirty-one operations were performed for multiple trunk involvement, recurrent disease, arteritis, infection, dissection, coarctation, or aneurysm. There were 122 bypass grafting procedures (98 ipsilateral, 24 contralateral) and 60 arterial transpositions. RESULTS: One death (0.5%) and 7 nonfatal strokes (3.8%) occurred, none in patients who were asymptomatic. Perioperative morbidity included four asymptomatic occlusions (2%), 6 myocardial infarctions (3%), 10 pulmonary complications (5%), and 2 graft infections (1%). Follow-up periods ranged from 1 to 190 months (mean, 53 +/- 5 months). Nineteen patients (10%) were lost to follow-up. Fifty-seven late deaths occurred, most from cardiac causes. Seven reconstructions necessitated late revision. The cumulative primary patency rate at 5 and 10 years was 91% +/- 2% and 82% +/- 5%, respectively. The survival rate at 5 years was 72% +/- 4% and at 10 years was 41% +/- 6%. The stroke-free survival rate was 92% +/- 2% at 5 years and 84% +/- 2% at 10 years. CONCLUSION: Cervical reconstruction of symptomatic and asymptomatic supra-aortic trunk lesions carries acceptable death and stroke rates and provides a long-term patient benefit. This should be the preferred approach for asymptomatic lesions and for patients with significant comorbidity because it carries less morbidity than direct transmediastinal aortic-based reconstruction.

A review of 100 consecutive reconstructions of the distal vertebral artery for embolic and hemodynamic disease.

PURPOSE: The aim of our study was to assess the outcome of distal vertebral artery (VA) reconstructions through a retrospective review conducted at a university-affiliated referral center. METHODS: One hundred consecutive distal VA reconstructions had been performed during a period of 14 years (98 patients) and included reversed saphenous vein bypass from the ipsilateral common, internal, or external carotid to the third portion of the VA at the C1-2 level (68 reconstructions) or the C0-1 level (4); transposition of the external carotid or its occipital branch to the VA (23); and transposition of the third portion of the VA onto the internal carotid artery (2). Other methods were used in 3 additional patients. Eighteen patients underwent concomitant carotid operations, and 1 patient underwent a concomitant subclavian transposition. Symptoms were present in 98% of patients and included vertebrobasilar ischemia (89%), vertebrobasilar plus hemispheric ischemia (7%), and hemispheric ischemia (2%). Two asymptomatic patients with bilateral carotid occlusions underwent operations to provide a single artery for cerebral perfusion (2%). Sixty-three lesions were atherosclerotic, 18 were dynamic bony compressions, and 14 were dissection, fibromuscular dysplasia, arteritis, or aneurysm. Five had miscellaneous anatomic indications. RESULTS: Stroke caused the four perioperative deaths that occurred. There was one occurrence of nonfatal hemispheric stroke. Routine postoperative arteriography identified 16 graft abnormalities; 11 patients underwent attempted revision. The introduction of the use of intraoperative angiography in 1990, halfway through the series, lowered the incidence of graft abnormalities from 28% to 4% and the incidence of perioperative death from 6% to 2%. Eighty-seven percent of patients had complete or significant resolution of symptoms. Follow-up ranged from 1 to 168 months (mean, 79 months). Ten patients were lost to follow-up. Twenty late deaths occurred; none were stroke related. Five reconstructions required late revision. The cumulative primary patency at 5 and 10 years was 75% +/- 6 and 70% +/- 7 (mean +/- SE), respectively; cumulative secondary patency was 84% +/- 5 and 80% +/- 6 at 5 and 10 years, respectively. Median survival was 107 months. CONCLUSIONS: Distal VA reconstruction provides excellent long-term patency and stroke protection. Intraoperative angiography is mandatory.

Carotid resection and replacement with autogenous arterial graft during operation for neck malignancy.

Carotid artery resection as part of the management of advanced head and neck cancers remains controversial. Since 1991, 30 patients have undergone resection of the carotid artery with immediate reconstruction using superficial femoral artery as replacement conduit. There was one stroke/death. Forty-three percent developed neck wound problems but no grafts failed or hemorrhaged. Mean follow-up was 20 months (3-76) and mean life expectancy was 16 months from the time of surgery. Fifty-eight percent were free of local recurrence at the time of death. There was a 35% disease-free survival rate at 2 years. These results compare favorably with alternative therapy including carotid ligation or shaving tumor from the carotid artery. Given the importance of cerebral perfusion and local tumor control we offer superficial femoral artery as a durable conduit for immediate extracranial carotid reconstruction in the often hostile environment associated with cancer resection in the neck.

Transthoracic repair of innominate and common carotid artery disease: immediate and long-term outcome for 100 consecutive surgical reconstructions.

PURPOSE: This is a review of 100 consecutive supraaortic trunk reconstructions (SAT) performed over 16 years. METHODS: There were eight innominate endarterectomies and 92 bypass procedures based on the thoracic aorta (n = 86) or proximal innominate artery (n = 6) in 98 patients 24 to 79 years of age. Indications included cerebrovascular ischemia in 83 and upper extremity ischemia in four. Thirteen patients were asymptomatic. An innominate lesion was bypassed in 78 cases. The left common carotid and left subclavian arteries required reconstruction in 38 and nine patients, respectively. Multiple trunks were reconstructed by direct bypass grafting in 35. Approach was via median sternotomy in 92, partial sternotomy in six, and left thoracotomy in two. Seven patients underwent concomitant cardiac surgery. RESULTS: Eight deaths and eight nonfatal strokes occurred, for a combined stroke/death rate of 16%. The operative mortality rate was 6% for SAT and 29% for SAT/cardiac operations. Perioperative complications included two asymptomatic graft occlusions, three nonfatal myocardial infarctions, seven significant pulmonary complications, three sternal wound infections, and one recurrent laryngeal nerve injury. Follow-up ranged from 1 to 184 months (mean, 51 +/- 4.8 months). Eight patients were lost to follow-up. Twenty-one late deaths occurred. Two SATs required late revision. The cumulative primary patency rates at 5 and 10 years were 94% +/- 3% and 88% +/- 6%, respectively. The stroke-free survival rates at 5 and 10 years were 87% +/- 4% and 81% +/- 7%, respectively. Patients who survived beyond 30 days had a median stroke-free life expectancy of 10 years, 7 months (SE, 6%). CONCLUSIONS: Direct reconstruction of complex symptomatic SAT lesions can be performed with acceptable death/stroke rates and with long-term patient benefit. Asymptomatic lesions in patients who have significant concomitant conditions should be managed with a less-morbid cervical or endovascular approach, even if long-term outcome of the latter is inferior.

End-diastolic volume. A better indicator of preload in the critically ill.

The relative value of pulmonary artery wedge pressure (PAWP) and right ventricular end-diastolic volume index (RVEDVI) as a reflection of the preload status of the critically ill was determined in 29 patients. Regression analysis of 131 hemodynamic studies demonstrated that cardiac index (CI) correlated better with RVEDVI (r = .61) than did PAWP (r = .42). Comparisons of PAWP and RVEDVI showed that possible misleading information concerning filling volume was provided by the PAWP at some time in 15 (52%) of these patients. In 15 patients given 22 fluid challenges, patients with a high PAWP (greater than or equal to 18 mm Hg) "responded" with a rise in CI more frequently than did patients with a low PAWP (less than 12 mm Hg). However, all eight patients with a RVEDVI less than 90 mL/m2 responded with a rise in CI, but all seven patients with a RVEDVI greater than or equal to 139 mL/m2 failed to respond. Thus, RVEDVI more accurately predicted preload recruitable increases in cardiac output.

Treatment of cerebral ischemia with Dextran-40 or Fluosol DA 20%.

A cat stroke model was used to evaluate the efficacy of Dextran-40 (DEX) or Fluosol-DA 20% (FDA) in the treatment of focal cerebral ischemia. The animals were assigned randomly to one of three treatment groups: control, isovolemic hemodilution with DEX or isovolemic hemodilution with FDA. The oxidation state of cytochrome aa3 was measured in-vivo using near infrared reflectance spectrophotometry. The cerebral edema was measured by magnetic resonance imaging (MRI). The MRI edema indices for the three groups revealed that the FDA group had less edema (p less than 0.05), approaching that of non-stroke controls. The relative oxidation state of aa3 for the DEX group declined both during and after hemodilution. At the ninth hour post stroke the FDA group was better (aa3 more oxidized. p less than 0.025). Changes in blood and plasma components were reflective of the extent of hemodilution. Whole blood viscosity analysis revealed a difference (p less than 0.05) at the lower shear rates comparing DEX to FDA with FDA being higher than DEX. Two animals in each of the groups were allowed to awaken at the end of the procedure for functional assessment. These observations suggest that hemodilution with FDA following stroke significantly reduces early post-ischemic cerebral edema, improves oxidation in the peri-infarct area and appears to minimize functional deficits.

Beneficial effects of isovolemic hemodilution using a perfluorocarbon emulsion in a stroke model.

In a clinically applicable cat stroke model, 16 purpose-bred adult animals were used to evaluate the beneficial effects of two treatment regimens: isovolemic hemodilution with either a perfluorocarbon emulsion or dextran 40 (a glucose polymer). Animals that received these treatment regimens were then compared with a control group of untreated animals. Focal cerebral infarctions were produced by transorbital ligation of the left middle cerebral artery. The randomly allocated treatment arms of the study were instituted 3 hours after ligation of the middle cerebral artery, thereby simulating a human clinical situation. In vivo mitochondrial metabolic activity of the peri-infarct cerebral tissue was continually assessed by means of a multiwavelength near-infrared spectrophotometer. This allowed measurement of cellular oxygenation at the cytochrome aa3 level, the terminal member of the cytochrome chain. Sequential proton-based magnetic resonance imaging was used to measure intracerebral water in vivo. Cardiac output, oxygen consumption/delivery, chemical, histologic, and rheologic parameters were also assessed. The data collected were analyzed by group means and standard statistical analyses, which revealed that the group treated with the perfluorocarbon emulsion had both less brain edema in the early post-infarct period (p less than 0.05), as well as a higher level of oxidation of cytochrome aa3 (p less than or equal to 0.025). This evidence supports the premise that isovolemic hemodilution with an oxygen-carrying hemodiluent may be beneficial in the treatment of ischemic strokes.

MRI quantitation of edema in focal cerebral ischemia in cats: correlation with cytochrome aa3 oxidation state.

1H MRI permits detection of edema in the brain. In a middle cerebral artery stroke model in the cat, we found a significant correlation between an edema index based on MRI and a sensitive metabolic index of ischemia, the in vivo oxidation status of mitochondrial cytochrome aa3 determined by near-infrared reflectance spectrophotometry (r = -0.70, alpha = 0.001). This result suggests that a simple, noninvasive study using MRI can provide an index of the extent of ischemic damage in an experimental acute stroke model.

Effect of antiarrhythmic drugs on defibrillation threshold: case report of an adverse effect of mexiletine and review of the literature.

Antiarrhythmic agents can influence defibrillation threshold (DFT). Basic research suggests that some class I drugs may have deleterious effects by raising defibrillation energy requirements. Evaluation of this problem in man has been limited to reports of patients who were more difficult to cardiovert or defibrillate after treatment with amiodarone and class IC agents. In the present report, mexiletine appeared to be the probable cause of an important elevation of DFT in a patient undergoing replacement of a malfunctioning automatic implantable cardioverter/defibrillator (AICD). This report and the accompanying literature review suggest that more information at both the basic and clinical levels is required. Retesting of device efficacy in terminating induced arrhythmia in the laboratory appears prudent in patients who require antiarrhythmic drug therapy subsequent to AICD implantation.

Ventricular fibrillation during coronary angiography: the precatheterization QT interval.

Ventricular fibrillation during coronary angiography is associated with contrast-induced changes in repolarization and thus pre-catheterization abnormalities could predispose to this event. We retrospectively examined angiograms, pre-catheterization electrocardiograms and records of 26 consecutive patients who had ventricular fibrillation during coronary angiography, and compared these patients to controls matched for age, sex, and left ventricular function. Diatrizoate meglumine was used as the angiographic contrast agent in all instances. Catheterization findings and the prevalence of prior myocardial infarction were similar in both groups. However, pre-catheterization QT intervals in the ventricular fibrillation group (0.43 +/- 0.05 sec) were significantly longer than in control patients (0.39 +/- 0.04 sec, P less than 0.005) as were their QT intervals corrected for heart rate (QTc) (0.47 +/- 0.04 vs 0.42 +/- 0.03 sec; P less than 0.001). Only seven of the 16 patients (44%) with ventricular fibrillation who had a precatheterization QTc greater than 0.44 sec had the arrhythmia during angiography of a critically stenosed (greater than 75%) coronary artery, whereas VF followed injection of critically stenosed vessels in eight of 10 (80%) of those with a normal QTc (p NS). After a follow-up period of 24 to 54 months (mean 39), two ventricular fibrillation patients have died (one suddenly), as compared to five in the control group (two suddenly) (p NS). Therefore, pre-catheterization QT prolongation was associated with ventricular fibrillation during coronary angiography, but ventricular fibrillation did not necessarily portend a worse long-term prognosis.