Because the origins of the biological safety profession are rooted in the control and prevention of laboratory-associated infections, the vocation focuses primarily on the safe handling of specimens within the laboratory. But in many cases, the specimens and samples handled in the lab are originally collected in the field where a broader set of possible exposure considerations may be present, each with varying degrees of controllability. The failure to adequately control the risks associated with collecting biological specimens in the field may result in illness or injury, and could have a direct impact on laboratory safety, if infectious specimens were packaged or transported inappropriately, for example. This study developed a web-based survey distributed to practicing biological safety professionals to determine the prevalence of and extent to which biological safety programs consider and evaluate field collection activities. In cases where such issues were considered, the data collected characterize the types of controls and methods of oversight at the institutional level that are employed. Sixty-one percent (61%) of the survey respondents indicated that research involving the field collection of biological specimens is conducted at their institutions. A majority (79%) of these field collection activities occur at academic institutions. Twenty-seven percent (27%) of respondents indicated that their safety committees do not consider issues related to biological specimens collected in the field, and only 25% with an oversight committee charged to review field collection protocols have generated a field research-specific risk assessment form to facilitate the assembly of pertinent information for a project risk assessment review. The results also indicated that most biosafety professionals (73% overall; 71% from institutions conducting field collection activities) have not been formally trained on the topic, but many (64% overall; 87% from institutions conducting field collection activities) indicated that training on field research safety issues would be helpful, and even more (71% overall; 93% from institutions conducting field collection activities) would consider participation in such a training course. Results obtained from this study can be used to develop a field research safety toolkit and associated training curricula specifically targeted to biological safety professionals.

The underlying causes of many reported laboratory associated infections (LAIs) are often not objectively identified and are generally attributed to poor laboratory technique or surface contamination in the laboratory. While training and practice can enhance laboratory technique, an objective evaluation of laboratory surface cleanliness is typically not performed. However, this is not the case for laboratories working with radioactive materials, where assessments for removable contamination are routinely performed. Similarly, industrial hygienists conduct surface sampling for metals and hexavalent chromium contamination, and the food-processing industry relies on adenosine triphosphate bioluminescence (ATB) swabs to confirm successful decontamination. The presence of ATB on surfaces is considered indicative of the presence of organic material that may harbor infectious organisms. In this study, baseline visual assessments, aerobic colony count (ACC), and ATB values were established for 10 active laboratories on five surface types cleaned with a fresh 10% dilution of 5.25% sodium hypochlorite. Baseline readings were used as controls against which to compare experimental readings collected in the laboratories over a 28-day period. Longitudinal results indicated a difference in failure percentages between the visual, ACC, and ATB methods such that failure percentages were greater (P < 0.001) for ATB. Note that the absence of surface contamination does not necessarily indicate that a LAI could not occur; however, the use of real-time ATB monitoring for surface contamination could possibly lead to a reduction in LAI events by facilitating the objective assessment of a possible key exposure route.

Serum levels of S-100B were investigated as a marker for infarct volume and response to treatment following acute ischemic stroke in rabbits. Following subselective angiography, rabbits (n=31) were embolized by injection of a 3-day-old blood clot (0.6x4.0-mm) into the internal carotid artery. Treatment began 1-hr post-embolization, groups included: Control (n=8, embolization only), tissue plasminogen activator (tPA, n=12, 0.9mg/kg), and perflutren lipid microbubbles with transcranial ultrasound (MB+US, n=11, MB at 0.16mg/kg, US at 1-MHz pulsed-wave, 0.8 W/cm2 for 1-hr). Serum S-100B levels were significantly increased (P<0.01) 24-hours following embolization in control (3.1-fold over baseline) and tPA (2.9-fold) groups, while treatment with MB+US resulted in an attenuated, non-significant (P=0.221) increase (1.6-fold). Twenty-four hour infarct volumes averaged 4.76%±1.16% for controls, 2.25%±0.95% for rabbits treated with tPA (P=0.32 vs. control), and 0.79%±0.99% for rabbits treated with MB+US (P=0.04 vs. control). Twenty-four hour concentrations of S-100B were positively correlated with infarct volume (r=0.59, P=0.0004).

Purpose: The New Zealand White rabbit (NZWR) serves an important role as an experimental model for vascular research, specifically in the area of stroke. Here the authors document vascular variations in the circle of Willis (COW).Materials and Methods: Subselective internal carotid digital subtraction angiography was performed in 100 NZWRs.Results: Important variations include hypoplasia in 36%, duplication of the middle cerebral artery in 29%, asymmetries of the posterior region in 19%, and multiple variations in 28%. The complete classical symmetric COW without significant variation is present in fewer than 30% of animals.Conclusions: With recognition of the variations, the NZWR becomes an improved research model.

Low-frequency ultrasound (US) enhances muscle and myocardial perfusion without lysis of the arterial obstruc-tion. We evaluated transcutaneous therapeutic ultrasound with tissue plasminogen activator (tPA) for decreas-es in cerebral infarct volume in rabbits with insoluble arterial occlusions. We hypothesized that US with tPA is effective at reducing infarcts without lysis of the arterial obstruction. New Zealand White rabbits (n=26; 5.2±0.08 kg) received angiography and three 700 to 900 μm embolic spheres were injected into the internal carotid artery occluding its branches. Treatment groups: control (n=10; embolized only), tPA alone (n=7), and tPA+US (n=9). Rabbits receiving US received pulsed-wave US (1-MHz, 0.8 W/cm2) over 1 hour. Rabbits administered tPA re-ceived intravenous tPA (0.9 mg/kg) over 1 hour. Rabbits were sacrificed 24 hours later and infarct volume was determined following staining with triphenyltetrazolium chloride. Immediate collateral flow was determined with digital subtraction angiography. Percent infarct volume was greater (P=0.035) for control (3.4±1.0%) than for tPA+US (0.9±1.0%) rabbits. Rabbits treated with tPA alone (2.0±1.1%) were intermediate and did not differ (P>0.29) from control and tPA+US rabbits. Collateral flow did not (P=0.93) influence infarct volume. Treatment with tPA+US decreases brain infarct volume in rabbits with permanent arterial occlusions, supporting flow stu-dies in muscle or myocardium.

Objectives:Increasing evidence confirms that microbubble (MB)-augmented ultrasound (US) thrombolysis enhances clot lysis with or without tissue plasminogen activator (tPA). Intracranial hemorrhage (ICH) is a major complication militating against tPA use in acute ischemic stroke. We quantified the incidence of ICH associated with tPA thrombolysis and MB + US therapy and compared infarct volumes in a rabbit model of acute ischemic stroke. Materials and Methods:Rabbits (n = 158) received a 1.0-mm clot, angiographically injected into the internal carotid artery causing infarcts. Rabbits were randomized to 6 test groups including (1) control (n = 50), embolized without therapy, (2) US (n = 18), (3) tPA only (n = 27), (4) tPA + US (n = 22), (5) MB + US (n = 27), and (6) tPA + MB + US (n = 14). US groups received pulsed wave US (1 MHz, 0.8 W/cm2) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Rabbits with MB received intravenous MB (0.16 mg/kg) given over 30 minutes. Rabbits were killed 24 hours later and infarct volume and incidence, location, and severity of ICH were determined by histology and pathologic examination. Results:Percentage of rabbits having ICH outside the infarct area was significantly decreased (P = 0.004) for MB + US (19%) rabbits compared with tPA + US (73%), US only (56%), tPA (48%), tPA + MB + US (36%), and control (36%) rabbits. Incidence and severity of ICH within the infarct did not differ (P > 0.39). Infarct volume was significantly greater (P = 0.002) for rabbits receiving US (0.97% ± 0.17%) than for MB + US (0.20% ± 0.14%), tPA + US (0.15% ± 0.16%), tPA (0.14% ± 0.14%), and tPA + MB + US (0.10% ± 20%) rabbits; these treatments collectively, excluding US only, differed (P = 0.03) from control (0.45% ± 0.10%). Conclusions:Treatment with MB + US after embolization decreased the incidence of ICH and efficacy was similar to tPA in reducing infarct volume.

Duration and extent of penumbra determine the window and brain volume in which interventions may save injured tissue after stroke. Understanding the penumbra in animals is necessary in order to design models that translate to effective clinical therapies. New Zealand white rabbits were embolized with aged autologous clot (n = 23) or insoluble microspheres (n = 21). To examine effects of treatment on penumbra, sphere-stroked animals were treated with 3 μm microbubbles plus ultrasound (n = 19). Rabbits were euthanized at 4 or 24 hr. Infarct volume was measured following triphenyltetrazolium chloride (TTC) staining of brain sections. Penumbra was visualized using immunostaining of pimonidazole injected fifteen minutes prior to euthanasia. Potentially reversible penumbra was present in 14.3% stroked rabbits at 4 hours and 15.7% at 24 hours after embolic stroke and represented up to 35% of total lost tissue. Intervention at up to 24 hours may benefit a significant patient population.

Background and Purpose— Microbubbles (MB) combined with ultrasound (US) have been shown to lyse clots without tissue-type plasminogen activator (tPA) both in vitro and in vivo. We evaluated sonothrombolysis with 3 types of MB using a rabbit embolic stroke model. Methods— New Zealand White rabbits (n=74) received internal carotid angiographic embolization of single 3-day-old cylindrical clots (0.6×4.0 mm). Groups included: (1) control (n=11) embolized without treatment; (2) tPA (n=20); (3) tPA+US (n=10); (4) perflutren lipid MB+US (n=16); (5) albumin 3 μm MB+US (n=8); and (6) tagged albumin 3 μm MB+US (n=9). Treatment began 1 hour postembolization. Ultrasound was pulsed-wave (1 MHz; 0.8 W/cm 2 ) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Lipid MB dose was intravenous (0.16 mg/kg) over 30 minutes. Dosage of 3 μm MB was 5×10 9 MB intravenously alone or tagged with eptifibatide and fibrin antibody over 30 minutes. Rabbits were euthanized at 24 hours. Infarct volume was determined using vital stains on brain sections. Hemorrhage was evaluated on hematoxylin and eosin sections. Results— Infarct volume percent was lower for rabbits treated with lipid MB+US (1.0%±0.6%; P =0.013), 3 μm MB+US (0.7%±0.9%; P =0.018), and tagged 3 μm MB+US (0.8%±0.8%; P =0.019) compared with controls (3.5%±0.8%). The 3 MB types collectively had lower infarct volumes ( P =0.0043) than controls. Infarct volume averaged 2.2%±0.6% and 1.7%±0.8% for rabbits treated with tPA alone and tPA+US, respectively ( P =nonsignificant). Conclusions— Sonothrombolysis without tPA using these MB is effective in decreasing infarct volumes. Study of human application and further MB technique development are justified.

Introduction:Tissue plasminogen activator (tPA) is the thrombolytic standard of care for acute ischemic stroke, but intracerebral hemorrhage (ICH) remains a common and devastating complication. We investigated using ultrasound (US) and microbubble (MB) techniques to reduce required tPA doses and to decrease ICH. Materials and Methods:Fresh blood clots (3–5 hours) were exposed in vitro to tPA (0.02 or 0.1 mg/mL) plus pulsed 1 MHz US (0.1 W/cm2), with or without 1.12 × 108/mL MBs (Definity or albumin/dextrose MBs [adMB]). Clot mass loss was measured to quantify thrombolysis. New Zealand white rabbits (n = 120) received one 3- to 5-hour clot angiographically delivered into the internal carotid artery. All had transcutaneous pulsed 1 MHz US (0.8 W/cm2) for 60 minutes and intravenous tPA (0.1–0.9 mg/kg) with or without Definity MBs (0.16 mL/mg/kg). After killing the animals, the brains were removed for histology 24 hours later. Results:In vitro, MBs (Definity or adMB) increased US-induced clot loss significantly, with or without tPA (P < 0.0001). At 0 and 0.02 mg/mL, tPA clot loss was greater with adMBs compared with Definity (P ≤ 0.05). With MB, the tPA dose was reduced 5-fold with good efficacy. In vivo, both Definity MB and tPA groups had less infarct volume compared with controls at P < 0.0183 and P = 0.0003, respectively. Definity MB+tPA reduces infarct volume compared with controls (P < 0.0001), and ICH incidence outside of strokes was significantly lower (P = 0.005) compared with no MB. However, infarct volume in Definity MB versus tPA was not different at P = 0.19. Conclusion:Combining tPAand MB yielded effective loss of clot with very low dose or even no dose tPA, and infarct volumes and ICH were reduced in acute strokes in rabbits. The ability of MBs to reduce tPA requirements may lead to lower rates of hemorrhage in human stroke treatment.

PURPOSE:Current rabbit stroke models often depend on symptoms as endpoints for embolization and produce wide variation in location, size, and severity of strokes. In a further refinement of an angiographic embolic stroke model, localized infarctions were correlated to neurologic deficits with the goal to create a rabbit model for long-term studies of therapies after stroke. MATERIALS AND METHODS:New Zealand White rabbits (4-5 kg; N = 71) had selective internal carotid artery (ICA) angiography and a single clot was injected. At 24 hours, neurologic assessment score (NAS) was measured on an 11-point scale (0, normal; 10, dead). Brains were removed and stained to identify stroke areas. All animals with single strokes (n = 31) were analyzed by specific brain structure involvement, and NAS values were correlated. RESULTS:Stroke incidence differed by location, with cortex, subcortical, and basal ganglia regions highest. The middle cerebral artery (MCA), at 52%, and anterior cerebral artery (ACA), at 29%, were most commonly involved, with the largest stroke volumes in the ACA distribution. Brainstem and cerebellum strokes had disproportionately severe neurologic deficits, scoring 2.25 +/- 1.0 on the NAS, which represented a significant (P < .02) difference versus cortex (0.5 +/- 0.2), subcortical (1.3 +/- 0.4), and basal ganglia (0.5 +/- 0.3), all in the frontal or parietal regions. CONCLUSIONS:MCA and ACA distributions included 81% of strokes. These sites were relatively silent (potentially allowing longer-term survival studies) whereas others in the posterior circulation produced disproportionately severe symptoms. Symptoms were not reliable indicators of stroke occurrence, and other endpoints such as imaging may be required. These are important steps toward refinement of the rabbit stroke model.

Introduction: Evidence suggests that low-frequency ultrasound (US) improves tissue perfusion in the rabbit ischemic limb and enhances myocardial perfusion even without removing the arterial obstruction. We evaluated US-facilitated thrombolysis, or sonothrombolysis, with microbubbles (MB) and tissue plasminogen activator (tPA) for decreases in infarct volume in rabbits with insoluble middle cerebral artery (MCA) occlusions. Hypothesis: Sonothrombolysis with MB or tPA is effective at reducing infarcts without lysis of arterial obstruction. Methods: New Zealand White rabbits (n = 34; 5.2±0.07 kg) received angiography and three embolic spheres (diameter = 700 to 900 μ m) were injected into the internal carotid artery occluding the MCA. Rabbits were randomly assigned to one of four groups. Control (n = 10) rabbits were embolized but received no US, tPA, or MB. Other groups were tPA (n = 7) without US, tPA+US (n = 9), and MB+US (n = 8). Treatment was initiated one hour following angiographic verification of occlusion. Rabbits with US received pulsed wave US (1 MHz at 0.8 W/cm 2 ) for 1 hour and rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Rabbits with MB received intravenous MB (0.16 mg/kg) over 30 minutes. Rabbits were sacrificed at 24 hours and infarct volume was determined using vital stains on brain sections. Results: Percent infarct volume averaged 3.4±0.9%, 2.0±1.1%, 1.8±1.0%, and 0.92±0.9% for control, tPA, MB+US, and tPA+US rabbits, respectively; infarct volume tended (P = 0.066) to be decreased for rabbits treated with tPA+US compared to controls. Infarct volume did not differ (P > 0.26) between controls and rabbits treated with tPA and MB+US. Conclusions: Combined treatment with tPA and US shows a trend towards decreasing brain infarct volume in rabbits with permanent MCA occlusion, but not to the extent found in muscle or myocardium at lower frequencies.

The rabbit serves as an attractive model for the study of ischemic stroke for many reasons. Despite its long-term use as an embolic stroke model, descriptions of anatomical variations in and close to the Circle of Willis (COW) are limited. These variations can impact distribution of emboli and strokes in this model and may contribute to variation in strokes designed to be uniform from animal to animal. Our objective is to describe the arterial variation in and close to the rabbit COW. Using 3 F catheter selection of the internal carotid artery, magnification digital subtraction angiography was performed on New Zealand white rabbits (N=100). Film and electronic images were studied. Vessels of the COW and its major branches were recorded as absent, attenuated, normal, enlarged, or exceptionally enlarged. Additionally, variations including unusual origination points, abnormal connections, or duplicate vessels, were documented. Overall, 29% of rabbits had no variations. However, attenuated vessels occurred in 36% of rabbits. Most attenuations were in anterior cerebral arteries (13%) and vertebral arteries (10%) with other sites widely distributed. Other main variations were duplicate middle cerebral arteries (DMCAs) and posterior cerebral-basilar artery variations. Unilateral DMCAs occurred in 27% and were bilateral in 2%. In 19% a posterior cerebral-basilar asymmetric variation consisted of one posterior cerebral artery that served as the origination of the contralateral posterior cerebral artery and the ipsilateral superior cerebellar artery. The contralateral superior cerebellar artery originated directly from the basilar artery. A vertebral artery was absent in 7%. Two or more variations occurred in 28%. Arterial variations in the COW of the rabbit are more common than the classic pattern which is seen in only 29%. Vessel attenuations accounted for most variation at 36%. Asymmetries in the vertebrobasilar system occur in 29%, and duplicate MCA origins occur in 27%. These common deviations from the norm may explain much of the variability seen in the rabbit when used as an embolic stroke model by either the internal carotid artery or vertebral approach.