Novel Human Acute Ischemic Stroke Blood Clot Analogs for In Vitro Thrombectomy Testing.

BACKGROUND AND PURPOSE: Previous studies have successfully created blood clot analogs for in vitro endovascular device testing using animal blood of various species. Blood components vary greatly among species; therefore, creating clot analogs from human blood is likely a more accurate representation of thrombi formed in the human vasculature. MATERIALS AND METHODS: Following approval from the Mayo Clinic institutional review board, human whole-blood and platelet donations were obtained from the blood transfusion service. Twelve clot analogs were created by combining different ratios of red blood cells + buffy coat, plasma, and platelets. Thrombin and calcium chloride were added to stimulate coagulation. Clot composition was assessed using histologic and immunohistochemical staining. To assess the similarities of mechanical properties to patient clots, 3 types of clot analogs (soft, elastic, and stiff) were selected for in vitro thrombectomy testing. RESULTS: The range of histopathologic compositions produced is representative of clots removed during thrombectomy procedures. The red blood cell composition ranged from 8.9% to 91.4%, and fibrin composition ranged from 3.1% to 53.4%. Platelets (CD42b) and von Willebrand Factor ranged from 0.5% to 47.1% and 1.0% to 63.4%, respectively. The soft clots had the highest first-pass effect and successful revascularization rates followed by the elastic and stiff clots. Distal embolization events were observed when clot ingestion could not be achieved, requiring device pullback. The incidence rate of distal embolization was the highest for the stiff clots due to the weak clot/device integration. CONCLUSIONS: Red blood cell-rich, fibrin-rich, and platelet-rich clot analogs that mimic clots retrieved from patients with acute ischemic stroke were created in vitro. Differing retrieval outcomes were confirmed using in vitro thrombectomy testing in a subset of clots.

The Relationship Between Cerebral Reperfusion And Regional Expression Of Matrix Metalloproteinase-9 In Rat Brain Following Focal Cerebral Ischemia.

We investigated the effect of full and partial mechanical reperfusion on MMP-9 expression in rat brain following middle cerebral artery occlusion, mimicking mechanical thrombectomy. Using percentage hemispheric lesion volume and oedema as measures, partial reperfusion reduced extent of brain damage caused by MCA occlusion, but the protective effect was less pronounced than with complete reperfusion. Using ELISA quantification in fresh frozen tissue, confirmed by immunofluorescence in perfusion fixed tissue, increased MMP-9 expression was observed in infarcted tissue. MMP-9 was increased in lesioned tissue of the anterior and posterior temporal cortex and underlying striatal tissue, but also the normal appearing frontal cortex. No significant increase in MMP-9 in the hippocampus was observed, nor in the unlesioned contralateral hemisphere. Both partial reperfusion and full reperfusion reduced the regional MMP expression significantly. The highest levels of MMP-9 were observed in lesioned brain regions in the non-reperfused group. MMP-9 expression was evident in microvessels and in neuronal cell bodies of affected tissue. This study shows that MMP-9 brain levels are reduced relative to the extent of reperfusion. These observations suggest targeting early increases in MMP-9 expression as a possible neuroprotective therapeutic strategy and highlight the rat MCA occlusion model as an ideal model in which to study candidate therapeutics.

Histological stroke clot analysis after thrombectomy: Technical aspects and recommendations.

The recent advent of endovascular procedures has created the unique opportunity to collect and analyze thrombi removed from cerebral arteries, instigating a novel subfield in stroke research. Insights into thrombus characteristics and composition could play an important role in ongoing efforts to improve acute ischemic stroke therapy. An increasing number of centers are collecting stroke thrombi. This paper aims at providing guiding information on thrombus handling, procedures, and analysis in order to facilitate and standardize this emerging research field.

Review of external referrals to a regional stroke centre: it is not just about thrombectomy.

AIMS: To determine the experience of a regional stroke referral centre of external referrals for endovascular thrombectomy (EVT) in patients with symptoms of acute ischaemic stroke (AIS) and large vessel occlusion (LVO). MATERIALS AND METHODS: Data were collected prospectively over two 4-month periods (2017-2018) on consecutive external referrals for EVT. Baseline demographics, imaging findings, and key time parameters were recorded. Reasons for not transferring patients and for not performing EVT were recorded. Key time intervals were calculated and compared between the transferred and non-transferred group with and without intracranial occlusion and between the transferred patients who underwent thrombectomy and those who did not. RESULTS: Two hundred and sixty-two patients were referred. Sixty-one percent (n=159) were accepted and transferred for treatment. Of those transferred, 86% (n=136) had EVT. Fourteen percent (n=23) were unsuitable for EVT on arrival due to no vessel occlusion (48% n=11), poor Alberta Stroke Program Early CT Score (ASPECTS)/established infarct (30%, n=7) haemorrhage (9%, n=2), and clinical recovery (13% n=3). One hundred and three patients (39%) were ineligible for EVT following phone discussion due to absence of intracranial occlusion (59%, n=61), low ASPECTS (22%, n=23), distal occlusion (4%, n=4), low/improving National Institutes of Health Stroke Scale (NIHSS; 10.7%, n=11), and poor modified Rankin Scale (mRS) at baseline (3%, n=3). Patients with LVO but not transferred had longer onset to hospital arrival time compared with those transferred 151.5 versus 91 minutes (p<0.005), with a trend also toward a longer door to CT/CTA 40 minutes versus 30 minutes (p=0.142). CONCLUSION: These data provide valuable insights into the service provision of a comprehensive stroke network. The present rates of EVT and futile transfers are modest compared to published data. Access to neuroradiology and specialised stroke assessment is crucial to optimise time to treatment.

A Standardized Aspiration-First Approach for Thrombectomy to Increase Speed and Improve Recanalization Rates.

BACKGROUND AND PURPOSE: Direct aspiration is a recognized technique for revascularization in large-vessel ischemic strokes. There is ongoing debate regarding its efficacy compared with stent retrievers. Every delay in achieving revascularization and a decrease in reperfusion rates reduces the likelihood of patients achieving functional independence. We propose a standardized setup technique for aspiration-first for all anterior circulation thrombectomy procedures for increasing speed and recanalization rates. MATERIALS AND METHODS: We analyzed 127 consecutive patients treated by a standardized approach to thrombectomy with an intention to perform aspiration-first compared with 127 consecutive patients treated with a stent retriever-first approach. Key time metrics evaluated included groin to first angiogram, first angiogram to reperfusion, groin to first reperfusion, and length of the procedure. The degree of successful recanalization (TICI 2b-3) and the number of passes were compared between the 2 groups. RESULTS: In 127 patients who underwent the standardized technique, the median time from groin puncture to first reperfusion was 18 minutes compared with 26 minutes (P < .001). The duration of the procedure was shorter compared with the stent retriever group (26 minutes in the aspiration first group versus 47 minutes, P < .001) and required fewer passes (mean, 2.4 versus 3.1; P < .05). A higher proportion of patients had a TICI score of 2b-3 in the aspiration-first group compared with stent retriever group (96.1% versus 85.8%, P < .005). CONCLUSIONS: Our study highlights the increasing speed and recanalization rates achieved with fewer passes in a standardized approach to thrombectomy with an intention to attempt aspiration-first. Any attempt to reduce revascularization time and increase successful recanalization should be used.

Animal Models of Focal Cerebral Ischaemia and Haemorrhagic Transformation: Considerations in Experimental Stroke Study Design.

BACKGROUND: Ischaemic stroke is often complicated with haemorrhage within the infarct zone or in a remote location especially when treated with intravenous thrombolysis and/or thrombectomy. While these early recanalisation treatments are highly effective, some of the benefit is lost because of haemorrhagic complications and consequential neurological deterioration of the patients. A number of mechanisms have been described that mediate the haemorrhagic changes and several agents have been tested in experimental models for inhibiting post-stroke haemorrhage. METHODS: Here, we review and discuss the small animal models of focal cerebral ischaemia and postischaemic stroke haemorrhagic transformation and how these models can best be utilised for developing further insights as well as potential treatment approaches for this serious clinical complication. RESULTS: The need to use appropriate animal models with relevant stroke risk factors to improve the clinical relevance and applicability of findings is becoming ever more apparent. Current focal ischaemia models can be adapted for the study of haemorrhagic transformation post-stroke. CONCLUSION: A number of factors can be added to the animal model design to increase the incidence and/or severity of haemorrhagic transformation post-ischaemic stroke, which can improve clinical relevance, aid the study of the pathophysiology and the future development of novel interventions.

Nerve growth factor-mediated inhibition of apoptosis post-caspase activation is due to removal of active caspase-3 in a lysosome-dependent manner.

Nerve growth factor (NGF) is well characterised as an important pro-survival factor in neuronal cells that can inhibit apoptotic cell death upstream of mitochondrial outer membrane permeabilisation. Here we addressed the question of whether NGF can also protect against apoptosis downstream of caspase activation. NGF treatment promoted a rapid reduction in the level of the p17 subunit of active caspase-3 in PC12 cells that had been induced to undergo apoptosis by various cytotoxins. The mechanism involved TrkA-dependent activation of extracellular signal-regulated kinase (ERK1/2) but not phosphatidylinositol 3-kinase (PI3K)/Akt, and de novo protein synthesis. Involvement of inhibitor of apoptosis proteins (IAPs) and proteasomal degradation were ruled out. In contrast, inhibition of lysosome function using chloroquine and concanamycin A reversed NGF-induced removal of p17. Moreover, in NGF-treated cells, active caspases were found to be localised to lysosomes. The involvement of macroautophagy and chaperone-mediated autophagy were ruled out. Taken together, these findings suggest an anti-apoptotic mechanism by which NGF induces removal of active caspase-3 in a lysosome-dependent manner.

Preliminary characterisation of an in vitro paradigm for the study of the delayed effects of organophosphorus compounds: hen embryo brain spheroids.

Organophosphate induced delayed neuropathy (OPIDN) has been studied extensively but the mechanisms of toxicity remain unclear. It is generally accepted that the inhibition and ageing (dealkylation) of the B-esterase neuropathy target esterase (NTE) is integral to axonal loss. At present, the only way of detecting compounds that induce OPIDN is the hen test, an animal model. In this study, we preliminary validated hen embryo brain spheroids (HEBS) for the study of organophosphate (OP) toxicity. Hen brain spheroids have been characterised previously, although they have never been fully optimised for OP testing. We optimised the levels of acetylcholine esterase (AChE) and neuropathy target esterase by adapting the culture technique and using chemically defined media. Spheroid cultures were maintained for 35 days and viability and enzyme levels were monitored over this time. Levels of AChE and NTE in this system remained stable over the 35 day period. Using transmission electron microscopy, we have shown synaptogenesis within HEBS earlier than previously suggested in spheroid culture. These studies indicate that HEBS may be useful for the study of OP-induced toxicity and that the long-term stability of the cultures makes it an ideal candidate for studying OPIDN.

The development and testing of the patient record pain management assessment tool.

The article reports a study whose purpose was to develop and test the Patient Record Pain Management Assessment Tool, an instrument to evaluate compliance with the American Pain Society's quality assurance standards on acute pain and cancer pain in chart documentation. Content validity, overall validity, and interrater reliability were all found to be acceptable. The instrument is therefore a useful tool for documenting the level of pain management practice in institutional settings.

Investigation of the actions and antagonist activity of some polyamine analogues in vivo.

1. The ability of three putative polyamine antagonists to antagonize behavioural changes induced by spermine was assessed. 2. Injection of an excitotoxic dose of spermine (100 microg, i.c.v.) in mice results in the development of a characteristic behavioural profile, which has two temporally distinct phases. The early events include clonic convulsions, and the later, more general excitation, includes tremor and culminates in the development of a fatal tonic convulsion. 3. Co-administration of arcaine (25 microg, i.c.v.) potentiated the early phase effects after spermine injection, but antagonized the development of spermine-induced tonic convulsions. A larger dose of arcaine (50 microg, i.c.v.) given alone resulted in the development of spermine-like body tremor and convulsions. It therefore appears that arcaine is not a pure polyamine antagonist in vivo, but may be a partial agonist. 4. Similarly, 1,10-diaminodecane appeared to act as a partial agonist in vivo, although it was less potent than arcaine. 5. In contrast, diethylenetriamine (DET) effectively inhibited the development of the early effects of spermine, but was ineffective against the spermine-induced CNS excitation and tonic convulsions. 6. It is concluded that none of the putative polyamine antagonists tested behaved as effective polyamine antagonists in vivo, although each produced some antagonism.

Comparison of various N-methyl-D-aspartate receptor antagonists in a model of short-term memory and on overt behaviour.

This study examined the effects on rat behaviour of antagonists acting at various sites on the N-methyl-D-aspartate (NMDA) receptor complex, i.e. the glutamate recognition site (CPP), ion channel (dizocilpine), glycine recognition site [(+)-HA-966] and the NR2B subunit-selective compound ifenprodil. Specifically, the effects of these agents were examined on working memory, assessed using the operant delayed match-to-position task (DMTP), and overt behaviour, assessed (a) in animals responding for food under a variable interval 20-s (VI20) schedule and (b) by spontaneous behaviour. Dizocilpine, CPP and (+)-HA-966 each reduced accuracy in the DMTP task independent of delay. At equivalent doses, changes in locomotor behaviour and VI20 responding were evident. In contrast, ifenprodil failed to impair accuracy in the DMTP task, even at doses that affected other performance measures and reduced VI20 responding. The relevance of these observations to neuroprotective and anticonvulsant doses of these compounds is considered.

Fatty acid ethyl esters in the blood as markers for ethanol intake.

OBJECTIVE: To determine the clinical utility of fatty acid ethyl esters (FAEEs) in the blood as a short-term confirmatory marker for ethanol intake and a longer-term marker for ethanol intake after ethanol is no longer detectable. DESIGN: Single-center controlled clinical trial and a blinded comparison involving 48 blood samples that were positive, negative, or equivocal for blood ethanol. PARTICIPANTS: Seven healthy subjects (4 men and 3 women, aged 21 to 23 years) participated in the clinical trial. Blood samples from participants for the blinded comparison portion of the study were numbered from 1 to 48 and not identified by name. INTERVENTION: The 7 healthy subjects ingested a known amount of ethanol at a fixed rate. The concentration of FAEEs in the blood after ethanol intake was determined for a period of up to 24 hours. There was no intervention in the blinded comparison study. MAIN OUTCOME MEASURES: In the clinical trial, a pharmacokinetic analysis of FAEE concentration in the blood after ethanol intake was completed for 7 individuals whose blood ethanol level was elevated from 25 to 35 mmol/L. In the blinded comparison, the 48 blood samples that were positive, negative, or equivocal for blood ethanol were analyzed for FAEE concentration. RESULTS: In the clinical trial, the disappearance of FAEEs from the blood followed a decay curve that initially resembled the decay curve for blood ethanol. However, because of a very slow secondary elimination phase, the FAEEs were found to persist in the blood for at least 24 hours after ethanol intake was completed. In the blinded comparison, all 20 samples that were positive for ethanol were positive for FAEEs, 7 of 7 samples equivocal for ethanol were positive for FAEEs, and 21 of 21 negative samples for ethanol were negative for FAEEs. CONCLUSIONS: Serum concentration of FAEEs can serve as an excellent short-term confirmatory test for ethanol intake as well as a longer-term marker of ethanol ingestion. Measurement of FAEEs in the blood may be a more sensitive indicator of ethanol ingestion than the measurement of blood ethanol .

Investigation of the involvement of the N-methyl-D-aspartate receptor macrocomplex in the development of spermine-induced CNS excitation in vivo.

1. The involvement of the N-methyl-D-aspartate (NMDA) receptor macrocomplex in the development of spermine-induced CNS excitation in vivo was investigated. 2. Injection of 100 micrograms of spermine into the left lateral cerebral ventricle of female Laca mice (20-25 g) resulted in the development of two distinct phases of CNS excitatory effects which were quantified by a scoring system. 3. The first phase effects occurred within minutes of injection and generally lasted for about 1 h. Most mice showed scratching of the upper body, frequent face washing and some mice developed clonic convulsions. By about 2 h after injection, the second phase of effects began to develop in the form of body tremor which worsened with time and culminated in fatal tonic convulsions, generally within 8 h of injection. 4. Pretreatment of the mice with dizocilpine (0.3 mg kg-1, i.p.) resulted in antagonism of the first phase of spermine-induced effects, but a higher dose (0.3 mg kg-1, (x2), i.p.) was necessary to inhibit the second phase effects. 5. Whereas the glutamate antagonist, 3-((R)-2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (D-CPP) (10, 20 mg kg-1, i.p.), the glycine antagonist 7-chlorokynurenate (10, 30, 50 nmol, i.c.v.), or the polyamine antagonist ifenprodil (30, 60 mg kg-1, i.p.) antagonized the first phase of effects produced by spermine, these agents given as monotherapy, were ineffective against the development of the second phase of effects. 6. Co-administration of ifenprodil with either D-CPP or 7-chlorokynurenate resulted in a dose-dependent antagonism of the development of the second phase of spermine-induced effects. 7. It is concluded that the development of the two temporally distinct phases of spermine-induced effects may be mediated by pharmacologically distinct mechanisms, although the results suggest that the NMDA receptor macrocomplex may be involved in both phases of effects. Furthermore, a moderate dose of D-CPP or 7-chlorokynurenate appears to enhance the inhibitory potential of ifenprodil in vivo.

Characterisation of a functional polyamine site on rat mast cells: association with a NMDA receptor macrocomplex.

Polyamines can modulate activation of N-methyl-D-aspartate (NMDA) receptors by binding to a specific polyamine site associated with a NMDA receptor macrocomplex. Polyamines induce histamine release from mast cells, although the mechanism had not been defined. We have examined whether spermine, a natural polyamine, and compound 48/80, regarded as a synthetic polyamine, activate mast cells by a polyamine site associated with a NMDA receptor macrocomplex. Spermine induced secretion of histamine from rat peritoneal mast cells and rat brain mast cells in a concentration-dependent manner. Rat peritoneal mast cells were used as a model system to explore the effects of NMDA antagonists on polyamine-induced histamine release. Ifenprodil, MK801 and arcaine inhibited histamine secretion from mast cells exposed to polyamines; the percentage inhibition was greater against spermine than compound 48/80. These data support the proposal that spermine (and possibly compound 48/80) induce histamine release from mast cells by interacting with a specific polyamine site on a NMDA receptor complex.

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography.

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70 +/- 3% (mean +/- S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsilyl (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individual FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.

Fatty acid ethyl esters are present in human serum after ethanol ingestion.

The aim of the study was to determine whether fatty acid ethyl esters, nonoxidative products of ethanol metabolism selectively present in organs damaged by ethanol abuse, are detectable in the serum after ethanol ingestion. Serum samples of hospital emergency room patients with positive (n = 32) and negative (n = 5) blood ethanol levels were assayed for fatty acid ethyl esters. In a separate study, five healthy subjects received an ethanol dose based on body weight mixed with fruit juice in a 1:2 ratio and administered by measured ingestion. Fatty acid ethyl esters were found in the serum of hospital emergency room patients with positive blood ethanol levels. The concentration of fatty acid ethyl esters in these patients correlated with the concentration of blood ethanol (r = 0.57; 95% confidence interval 0.28 to 0.77; P = 0.0002). In the controlled ethanol ingestion study with five healthy subjects, it was also determined that the serum fatty acid ethyl ester concentration began to decrease within 2 h of the time ethanol ingestion had been stopped. The fatty acid ethyl esters in the serum were bound to lipoprotein and albumin, and there was a higher percentage of saturated fatty acids in the FAEE pool than in the serum free fatty acid and triglyceride pools. These studies indicate that fatty acid ethyl esters, which have been implicated as mediators of ethanol-induced organ toxicity, are present in serum after ethanol ingestion.

DESEMO seats for young children with cerebral palsy.

Twelve children with cerebral palsy ages 18 months to eight years received custom-molded DESEMO seats. All had markedly abnormal motor function manifested in spastic, athetoid, or mixed patterns of movement. None sat independently or were expected to gain such function. Three had minimal head control, and the remaining nine had fair head control. All were inadequately positioned in commercially available seats. The custom-molded DESEMO seat was clinically evaluated with regard to its ability to produce overall relaxation, assist head control, enhance feeding, improve upper extremity function, and prevent deformities. Also assessed were parents' acceptance of the device, cost, cosmesis, convenience, and its capacity to accommodate growth. Seven children utilized the seat as an insert for a motorized wheelchair. The secure positioning provided by this device permitted operation of a standard hand operated joystick control. Two seats were used as floor sitters, allowing the children to participate in peer group activities. Feeding improved dramatically with two children, as evidenced by weight gain and decreased feeding time. Three children with kyphotic spinal deformities showed no improvement or progression of their curves despite the seating device. The positioning device assisted head control for 10 children but failed to enhance head control in the remaining two. The overall cost of the device, including orthotist and physical therapist time, is $640.00 (1985). The average growth potential of the seat is six to nine months.