Endotheliitis and cytokine storm as a mechanism of clot formation in COVID-19 ischemic stroke patients: A histopathologic study of retrieved clots.

BACKGROUND: Studies during the COVID-19 pandemic have demonstrated an association between COVID-19 virus infection and the development of acute ischemic stroke, particularly large vessel occlusion (LVO). Studying the characteristics and immunohistochemistry of retrieved stroke emboli during mechanical thrombectomy for LVO may offer insights into the pathogenesis of LVO in COVID-19 patients. We examined retrieved COVID-19 emboli from the STRIP, EXCELLENT, and RESTORE registries and compared their characteristics to a control group. METHODS: We identified COVID-positive LVO patients from the STRIP, RESTORE, and EXCELLENT studies who underwent mechanical thrombectomy. These patients were matched to a control group controlling for stroke etiology based on Trial of Org 10172 in Acute Stroke Treatment criteria. All clots were stained with Martius Scarlet Blue (MSB) along with immunohistochemistry for interleukin-6 (IL-6), C-reactive protein (CRP), von Willebrand factor (vWF), CD66b, fibrinogen, and citrullinated Histone H3. Clot composition was compared between groups. RESULTS: Nineteen COVID-19-positive patients and 38 controls were included. COVID-19-positive patients had a significantly higher percentage of CRP and vWF. There was no difference in IL-6, fibrin, CD66b, or citrullinated Histone H3 between groups. Based on MSB staining, there was no statistically significant difference regarding the percentage of red blood cells, white blood cells, fibrin, and platelets. CONCLUSIONS: Our study found higher concentrations of CRP and vWF in retrieved clots of COVID-19-positive stroke patients compared to COVID-19-negative controls. These findings support the potential role of systemic inflammation as indicated by elevated CRP and endothelial injury as indicated by elevated vWF as precipitating factors in thrombus development in these patients.

Effect of Empiric Treatment of Asymptomatic Bacteriuria in Neurosurgical Trauma Patients on Surgical Site and Clostridium difficile Infection.

BACKGROUND: Although empiric treatment of urinary tract colonized patients remains a frequent practice in neurosurgery, the value of this practice remains debatable. OBJECTIVE: To analyze the effect of screening and treatment of bacteriuria on surgical site infections, incidence of Clostridium difficile, and mortality in neurosurgical trauma patients. METHODS: Database queries and direct patient chart reviews were used to gather patient chart data. T-tests, chi-square tests, binary logistic regressions, and propensity matched cohorts comparisons were performed. RESULTS: A total of 3563 admitted neurosurgical trauma patients were identified over an 8 yr period (1524 cranial, 1778 spinal, and 261 combined craniospinal diagnoses). Nine hundred ninety-one patients underwent an operative neurosurgical procedure. Urinalysis was significantly associated with antibiotics exposure in both operative and nonoperative patients (P < .001). Operative patients treated with empiric antibiotics did not have a reduced risk of wound infection (P = .21), including in a propensity matched cohort (P = .52). Patients treated with empiric antibiotics had significantly increased rates of C. difficile infection (P < .001). At last follow-up, neurosurgical trauma patients that developed C. difficile had an increased risk of death (P < .005); antibiotic exposure and death were also significantly associated (P = .018). The association of C. difficile with empiric antibiotics remained significant in a propensity-matched cohort (P = .0024). CONCLUSION: The routine use of urinalysis and empiric urinary antibiotics for bacteriuria in neurosurgical trauma patients without urinary symptoms increases risk of exposure to antibiotics does not decrease rates of wound infection, and is associated with increased rates of C. difficile infection and death.

Paradoxical cerebral air embolism causing large vessel occlusion treated with endovascular aspiration.

Cerebral air embolism is a dreaded complication of invasive medical procedures. The mainstay of therapy for patients with cerebral air embolism has been hyperbaric oxygen therapy, high flow oxygen therapy, and anticonvulsants. We present a novel therapeutic approach for treatment of cerebral air embolism causing large vessel occlusion, using endovascular aspiration. Our patient developed a cerebral air embolism following sclerotherapy for varicose veins. This caused near total occlusion of the superior division of the M2 segment of the right middle cerebral artery. Symptoms included unilateral paralysis, unintelligible speech, and hemianopia; National Institutes of Health Stroke Scale (NIHSS) on presentation was 16. The air embolism was treated using a distal aspiration technique. Angiography following aspiration showed Thrombolysis in Cerebral Infarction 2B reperfusion. Following aspiration, the patient was re-examined; NIHSS at that time was 4. At 1 month follow-up, the modified Rankin Scale score was 1 and NIHSS was 1. Treatment of cerebral air embolism is discussed.

Insulin-like growth factor binding protein 7 and tissue inhibitor of metalloproteinases-2: differential expression and secretion in human kidney tubule cells.

We have characterized the expression and secretion of the acute kidney injury (AKI) biomarkers insulin-like growth factor binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2) in human kidney epithelial cells in primary cell culture and tissue. We established cell culture model systems of primary kidney cells of proximal and distal tubule origin and observed that both proteins are indeed expressed and secreted in both tubule cell types in vitro. However, TIMP-2 is both expressed and secreted preferentially by cells of distal tubule origin, while IGFBP7 is equally expressed across tubule cell types yet preferentially secreted by cells of proximal tubule origin. In human kidney tissue, strong staining of IGFBP7 was seen in the luminal brush-border region of a subset of proximal tubule cells, and TIMP-2 stained intracellularly in distal tubules. Additionally, while some tubular colocalization of both biomarkers was identified with the injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, both biomarkers could also be seen alone, suggesting the possibility for differential mechanistic and/or temporal profiles of regulation of these early AKI biomarkers from known markers of injury. Last, an in vitro model of ischemia-reperfusion demonstrated enhancement of secretion of both markers early after reperfusion. This work provides a rationale for further investigation of these markers for their potential role in the pathogenesis of acute kidney injury.

Paradoxical cerebral air embolism causing large vessel occlusion treated with endovascular aspiration.

Cerebral air embolism is a dreaded complication of invasive medical procedures. The mainstay of therapy for patients with cerebral air embolism has been hyperbaric oxygen therapy, high flow oxygen therapy, and anticonvulsants. We present a novel therapeutic approach for treatment of cerebral air embolism causing large vessel occlusion, using endovascular aspiration. Our patient developed a cerebral air embolism following sclerotherapy for varicose veins. This caused near total occlusion of the superior division of the M2 segment of the right middle cerebral artery. Symptoms included unilateral paralysis, unintelligible speech, and hemianopia; National Institutes of Health Stroke Scale (NIHSS) on presentation was 16. The air embolism was treated using a distal aspiration technique. Angiography following aspiration showed Thrombolysis in Cerebral Infarction 2B reperfusion. Following aspiration, the patient was re-examined; NIHSS at that time was 4. At 1 month follow-up, the modified Rankin Scale score was 1 and NIHSS was 1. Treatment of cerebral air embolism is discussed.

The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine.

Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.

Comparison of Intraoperative Indocyanine Green Angiography and Digital Subtraction Angiography for Clipping of Intracranial Aneurysms.

BACKGROUND: Residual aneurysm after microsurgical clipping carries a risk of aneurysm growth and rupture. Digital subtraction angiography (DSA) remains the standard to determine the adequacy of clipping. Intraoperative indocyanine green (ICG) angiography is increasingly utilized to confirm optimal clip positioning across the neck and to evaluate the adjacent vasculature. OBJECTIVE: We evaluated the correlation between ICG and DSA in clipped intracranial aneurysms. METHODS: A retrospective study of patients who underwent craniotomy and microsurgical clipping of intracranial aneurysms with ICG for 2 years. Patient characteristics, presentation details, operative reports, and pre- and postclipping angiographic images were reviewed to determine the adequacy of the clipping. RESULTS: Forty-seven patients underwent clipping with ICG and postoperative DSA: 57 aneurysms were clipped; 23 patients (48.9%) presented with subarachnoid hemorrhage. Nine aneurysms demonstrated a residual on DSA not identified on ICG (residual sizes ranged from 0.5 to 4.3 mm; average size: 1.8 mm). Postoperative DSA demonstrated no branch occlusions. CONCLUSION: Intraoperative ICG is useful in the clipping of intracranial aneurysms to ensure a gross patency of branch vessels; however, the presence of residual aneurysms and subtle changes in flow in branch vessels is best seen by DSA. This has important clinical implications with regard to follow-up imaging and surgical/endovascular management.

Outcomes of a novel minimalist approach for the treatment of cubital tunnel syndrome.

We describe a minimalist approach to perform in situ decompression of the ulnar nerve. Our technique employs a unique small skin incision strategically placed to minimize postoperative scarring over the ulnar nerve and potentially decrease the risk of iatrogenic injury to the medial antebrachial cutaneous nerve. We retrospectively report the outcome of patients who have undergone this procedure at our institution, the Michael E. DeBakey Veterans Affairs Medical Center, from January 1 2007 through November 29 2010. All individuals underwent in situ decompression via the previously described minimalist approach. Outcome variables were Louisiana State University Medical Center (LSU) ulnar neuropathy grade, patient satisfaction, subjective improvement, complications and re-operation rate. A total of 44 procedures were performed in this cohort of 41 patients. Overall, patients' postoperative LSU grades showed a statistically significant improvement (p=0.0019) compared to preoperative grades. Improvement of at least one grade in the LSU scale was observed in 50% of the procedures with a preoperative grade of four or less. Overall procedure satisfaction rate was 88% (39 of 44) with 70% (31 of 44) of the procedures resulting in improvement of symptoms. There were no intraoperative or postoperative complications. One patient required re-operation due to failure of neurological improvement. Our minimalistic approach to perform in situ decompression of the ulnar nerve at the cubital tunnel is both safe and effective. We observed a statistically significant improvement in LSU ulnar neuropathy grades and a success rate comparable to those reported for other more extensive surgical techniques while providing the benefit of a smaller incision, less scarring, decreased risk of iatrogenic nerve injury and minimal complications.

Rupture of giant vertebrobasilar aneurysm following flow diversion: mechanical stretch as a potential mechanism for early aneurysm rupture.

A patient with a giant symptomatic vertebrobasilar aneurysm was treated by endoscopic third ventriculostomy for obstructive hydrocephalus followed by treatment of the aneurysm by flow diversion using a Pipeline Embolization Device. After an uneventful procedure and initial periprocedural period, the patient experienced an unexpected fatal subarachnoid hemorrhage 1 week later. Autopsy demonstrated extensive subarachnoid hemorrhage and aneurysm rupture (linear whole wall rupture). The patent Pipeline Embolization Device was in its intended location, as was the persistent coil occlusion of the distal left vertebral artery. The aneurysm appeared to rupture in a linear manner and contained a thick large expansile clot that seemed to disrupt or rupture the thin aneurysm wall directly opposite the basilar artery/Pipeline Embolization Device. We feel the pattern of aneurysm rupture in our patient supports the idea that the combination of flow diversion and the resulting growing intra-aneurysmal thrombus can create a mechanical force with the potential to cause aneurysm rupture.

Distal aspiration with retrievable stent assisted thrombectomy for the treatment of acute ischemic stroke.

OBJECTIVE: Flexible large lumen aspiration catheters and stent retrievers have recently become available in the USA for the revascularization of large vessel occlusions presenting within the context of acute ischemic stroke (AIS). We describe a multicenter experience using a combined aspiration and stent retrieval technique for thrombectomy. DESIGN: A retrospective analysis to identify patients receiving combined manual aspiration and stent retrieval for treatment of AIS between August 2012 and April 2013 at six high volume stroke centers was conducted. Outcome variables, including recanalization rate, post-treatment National Institutes of Health Stroke Scale (NIHSS) score, symptomatic intracranial hemorrhage, discharge 90 day modified Rankin Scale (mRS) score, and mortality were evaluated. RESULTS: 105 patients were found that met the inclusion criteria for this retrospective study. Successful recanalization (Thrombolysis in Cerebral Infarction score 2B) was achieved in 92 (88%) of these patients. 44% of patients had favorable (mRS score 0-2) outcomes at 90 days. There were five (4.8%) symptomatic intracerebral hemorrhages and three procedure related deaths (2.9%). CONCLUSIONS: Mechanical thrombectomy utilizing combined manual aspiration with a stent retriever is an effective and safe strategy for endovascular recanalization of large vessel occlusions presenting within the context of AIS.

Rupture of giant vertebrobasilar aneurysm following flow diversion: mechanical stretch as a potential mechanism for early aneurysm rupture.

A patient with a giant symptomatic vertebrobasilar aneurysm was treated by endoscopic third ventriculostomy for obstructive hydrocephalus followed by treatment of the aneurysm by flow diversion using a Pipeline Embolization Device. After an uneventful procedure and initial periprocedural period, the patient experienced an unexpected fatal subarachnoid hemorrhage 1 week later. Autopsy demonstrated extensive subarachnoid hemorrhage and aneurysm rupture (linear whole wall rupture). The patent Pipeline Embolization Device was in its intended location, as was the persistent coil occlusion of the distal left vertebral artery. The aneurysm appeared to rupture in a linear manner and contained a thick large expansile clot that seemed to disrupt or rupture the thin aneurysm wall directly opposite the basilar artery/Pipeline Embolization Device. We feel the pattern of aneurysm rupture in our patient supports the idea that the combination of flow diversion and the resulting growing intra-aneurysmal thrombus can create a mechanical force with the potential to cause aneurysm rupture.

Analyzing cell physiology in C. elegans with fluorescent ratiometric reporters.

Ratiometric fluorescent reporters have recently emerged a new technique to non-invasively measure aspects of cell physiology such as redox status, calcium levels, energy production, and NADH levels. These reporters consist of either a single or pair of fluorophores along with specific modifications, such as the addition of a protein domain which binds to a metabolite of interest, thereby producing gradual alterations in fluorescence in response to changes in the measured parameter. Measurement of the changes in fluorescence produces a quantitative read-out of the cellular environment. While these reporters were initially developed to easily visualize and track changes in cultured cells, several groups have adapted these reporters to use in Caenorhabditis elegans which opens a new avenue through which to explore cell physiology during development or aging, in response to changes in external environment, or in response to genetic manipulation. These reporters have the advantage of being easily targeted to any part of the worm, and because C. elegans is transparent both the reporters and changes in their fluorescence can be clearly observed in vivo. Here we discuss the application of ratiometric reporters to C. elegans, and outline a method to quantitatively measure changes in intracellular peroxide levels using the HyPer ratiometric reporter. However, these principles can be applied to alternate ratiometric reporters which are designed to measure either other chemical species or other cellular parameters.

Imaging lysosomal enzyme activity in live cells using self-quenched substrates.

Endocytosis, the internalization and transport of extracellular cargo, is an essential cellular process. The ultimate step in endocytosis is the intracellular degradation of extracellular cargo for use by the cell. While live cell imaging and single particle tracking have been well-utilized to study the internalization and transport of cargo, the final degradation step has required separate biochemical assays. We describe the use of self-quenched endocytic cargo to image the intracellular transport and degradation of endocytic cargo directly in live cells. We first outline the fluorescent labeling and quantification of two common endocytic cargos: a protein, bovine serum albumin, and a lipid nanoparticle, low-density lipoprotein. In vitro measurements confirm that self-quenching is a function of the number of fluorophores bound to the protein or particle and that recovery of the fluorescent signal occurs in response to enzymatic degradation. We then use confocal fluorescence microscopy and flow cytometry to demonstrate the use of self-quenched bovine serum albumin with standard fluorescence techniques. Using live cell imaging and single particle tracking, we find that the degradation of bovine serum albumin occurs in an endo-lysosomal vesicle that is positive for LAMP1.

Fluorescent coumarin thiols measure biological redox couples.

In this report we present a new chemical probe, 3-HTC, that can reversibly and ratiometrically measure the thiol-disulfide equilibrium of biological systems. 3-HTC is composed of a coumarin that has a thiolate directly conjugated to its extended aromatic π system while formation of a disulfide attenuates this conjugation. The fluorescence and absorption properties of 3-HTC are therefore very sensitive to the redox state of its thiol. 3-HTC reacts reversibly with thiols and disulfides enabling its use to measure dynamic GSH/GSSH ratios in vitro as well as to monitor the reversible redox status of whole cell lysates.

Endo-lysosomal vesicles positive for Rab7 and LAMP1 are terminal vesicles for the transport of dextran.

The endo-lysosomal pathway is essential for intracellular transport and the degradation of extracellular cargo. The relationship between three populations of endo-lysosomal vesicles--Rab7-positive, LAMP1-positive, and both Rab7- and LAMP1-postive--was probed with fluorescence microscopy and single particle tracking. Of specific interest was determining if these vesicles were intermediate or terminal vesicles in the transport of extracellular cargo. We find that the major organelle in the endo-lysosomal pathway, both in terms of population and cargo transport, is positive for Rab7 and LAMP1. Dextran, a fluid phase cargo, shifts from localization within all three populations of vesicles at 30 minutes and 1 hour to primarily LAMP1- and Rab7/LAMP1-vesicles at longer times. This demonstrates that LAMP1- and Rab7/LAMP1-vesicles are terminal vesicles in the endo-lysosomal pathway. We tested two possible mechanisms for this distribution of cargo, delivery to mannose 6-phosphate receptor (M6PR)-negative vesicles and the fusion dynamics of individual vesicles. We find no correlation with M6PR but do find that Rab7-vesicles undergo significantly fewer fusion events than LAMP1- or Rab7/LAMP1-vesicles suggesting that the distribution of fluid phase cargo is driven by vesicle dynamics.

Clinical outcome of leukemia patients with intracranial hemorrhage. Clinical article.

OBJECT: Intracranial hemorrhage (ICH) is a frequent complication found in leukemia patients with thrombocytopenia. At the University of Texas MD Anderson Cancer Center, when a leukemia patient is found to have ICH, a platelet transfusion is generally recommended until 50,000/µl is reached. The authors examine the feasibility and outcome of their intervention strategy in this study. METHODS: Records were reviewed from 76 consecutive leukemia patients with newly diagnosed ICH at the University of Texas MD Anderson Cancer Center from January 1, 2007, to December 31, 2009. Variables of interest included age, platelet count at presentation, leukemia subtype, history of trauma, Glasgow Coma Scale score at presentation, whether the 50,000/µl goal was reached after transfusion, and whether the patient was a transfusion responder (platelet count increase > 2000/µl/unit transfused). Outcome parameters were mortality rates at 72 hours and 30 days and imaging-documented hemorrhage progression. RESULTS: Thrombocytopenia was prevalent at the time of presentation (68 of 76 patients had platelet levels < 50,000/µl at presentation). Despite an aggressive transfusion protocol, only 24 patients reached the 50,000/µl target after an average of 16 units of transfusion. Death due to ICH occurred in 15 patients within the first 72 hours (mortality rate 19.7%). Death correlated with the presenting Glasgow Coma Scale score (p = 0.0075) but not with other transfusion-related parameters. A significant mortality rate was again observed after 30 days (32.7%). The 30-day mortality rate, however, was largely attributable to non-ICH related causes and correlated with patient age (p = 0.032) and whether the patient was a transfusion responder (p = 0.022). Reaching and maintaining a platelet count > 50,000/µl did not positively correlate with the 30-day mortality rate (p = 0.392 and 0.475, respectively). CONCLUSIONS: Platelet transfusion in the setting of ICH in leukemia patients is undoubtedly necessary, but whether the transfusion threshold should be 50,000/µl remains unclear. Factors other than thrombocytopenia likely contribute to the overall poor prognosis.