Chemical inhibitors of phagosome-lysosome fusion in cultured macrophages also inhibit saltatory lysosomal movements. A combined microscopic and computer study.

The effects on lysosomal movements produced by the weak base ammonium chloride and by a representative polyanion poly-D-glutamic acid (PGA), previously reported to inhibit phagosome-lysosome (P-L) fusion, have been studied in cultured mouse macrophages using direct visual phase-contrast microscopy, a previously described (1, 3, 7) fluorescence assay of fusion, and computer analysis techniques. Treatment of the macrophages with 5-10 mM NH4Cl for 0.5-2 h or with 100 micrograms PGA/ml for 5 d caused a striking inhibition of saltatory lysosomal movements, as well as the expected inhibition of P-L fusion. Two other anionic fusion inhibitors tested, dextran sulphate and suramin, inhibited movements similarly. Removal of the NH4Cl from the cell medium reversed the lysosomal stasis and restored P-L fusion. Computer analyses of changes in lysosomal positions in treated and untreated macrophages during 2, 10, and 30-s intervals, using data from photomicrographs, computer graphics, and quantitative nearest-neighbour techniques developed for this purpose, supported the qualitative visual observation of the inhibition of lysosomal movements by the fusion inhibitors NH4Cl and PGA. Over the chosen intervals, from 80 to 96% of the lysosomes could be paired within 1 micron of each other in the NH4Cl- and PGA-treated cells in comparison with 50-70% in normal cells. The differences between the drug-treated and normal cells were highly significant. In an analogous system, the lysosomal stasis induced by hypertonic sucrose was examined and it was observed that P-L fusion too was inhibited. Both effects were reversible. We conclude that inhibition of P-L fusion and of lysosomal movement are associated. We suggest a causal relationship between these changes, namely, that the lysosomotropic inhibitors of fusion under study produce their effects largely, though perhaps not exclusively, by reducing saltatory lysosomal motion and consequently periphagosomal assembly, rather than directly and independently on P-L contact or on the fusion process itself. The possibility is raised that microtubules may be involved in the effector mechanism of these modulations.

The effects of dizocilpine maleate (MK-801), an antagonist of the N-methyl-D-aspartate receptor, on neurologic recovery and histopathology following complete cerebral ischemia in primates.

The present study was designed to determine if the noncompetitive excitatory amino acid antagonist, dizocilpine maleate, when administered after a 17 min period of complete cerebral ischemia in primates, would improve postischemic neurologic function and hippocampal histopathologic outcome when compared to placebo-treated animals. Ten pigtail monkeys were anesthetized and subjected to complete cerebral ischemia using an established neck tourniquet model. Five minutes postischemia, five monkeys received dizocilpine 300 micrograms/kg i.v. over 5 min, followed by an infusion of 150 micrograms/kg/h for 10 h. This produced plasma levels of the drug in excess of 30 ng/ml for the duration of the infusion. An additional five monkeys were treated with an identical volume of saline placebo. All monkeys received intensive care for the initial 24 to 48 h postischemia. At 96 h postischemia, there was no significant difference in neurologic function between the two groups (p = 0.53, with the placebo group having the numerically better outcome). There also was no significant difference between hippocampal histopathology scores between dizocilpine and placebo-treated monkeys. The authors conclude that dizocilpine is not an efficacious therapy in the treatment of neurologic injury that occurs following complete cerebral ischemia in this primate model.

Awake craniotomy for aggressive resection of primary gliomas located in eloquent brain.

OBJECTIVE: To determine with intraoperative neurologic and language examinations the maximal tumor resection achievable with acceptable postoperative neurologic dysfunction in patients undergoing awake stereotactic glial tumor resection in eloquent regions of the brain. PATIENTS AND METHODS: Between October 1995 and December 2000, 65 patients underwent frameless stereotactic resection of glial tumors located in functioning tissue. During the resection, continuous examinations by a neurologist and speech pathologist were performed. The goal of surgery was to resect the maximum neurologically permissible tumor volume defined on preoperative T2 imaging. Tumor resection was stopped at the onset of neurologic dysfunction. Novel segmentation software was used to measure tumor cytoreduction based on pre- and postoperative magnetic resonance imaging. All patients underwent 3-month postoperative neurologic examinations to determine functional outcomes. RESULTS: The cortical and subcortical white matter tracts at risk for injury were the left frontal operculum in 15 patients, the central lobule in 38, the insula in 11, and the left angular gyrus in 1. Thirty-four (52%) had a greater than 90% reduction in T2 signal postoperatively. In 26 patients thought to have low-grade tumors based on preoperative imaging, 12 proved to have grade 3 gliomas. Forty-eight patients (74%) developed intraoperative deficits; 34 (71%) recovered to a modified Rankin grade of 0 or 1 at 3 months postoperatively, 11 (23%) achieved a modified Rankin grade of 2, and 3 patients (6%) achieved a modified Rankin grade of 3 or 4 at 3-month follow-up. There was no operative mortality; 17 patients (26%) died from tumor progression during the follow-up period. CONCLUSIONS: Combining frameless computer-guided stereotaxis with cortical stimulation and repetitive neurologic and language assessments facilitates tumor resection in functioning brain regions. Resecting tumor until the onset of neurologic deficits allows for a good functional recovery. Imaging software can objectively and accurately measure preoperative and postoperative tumor volumes.

Skeletal muscle force and actomyosin ATPase activity reduced by nitric oxide donor.

Nitric oxide (NO) may exert direct effects on actin-myosin cross-bridge cycling by modulating critical thiols on the myosin head. In the present study, the effects of the NO donor sodium nitroprusside (SNP; 100 microM to 10 mM) on mechanical properties and actomyosin adenosinetriphosphatase (ATPase) activity of single permeabilized muscle fibers from the rabbit psoas muscle were determined. The effects of N-ethylmaleimide (NEM; 5-250 microM), a thiol-specific alkylating reagent, on mechanical properties of single fibers were also evaluated. Both NEM (>/=25 microM) and SNP (>/=1 mM) significantly inhibited isometric force and actomyosin ATPase activity. The unloaded shortening velocity of SNP-treated single fibers was decreased, but to a lesser extent, suggesting that SNP effects on isometric force and actomyosin ATPase were largely due to decreased cross-bridge recruitment. The calcium sensitivity of SNP-treated single fibers was also decreased. The effects of SNP, but not NEM, on force and actomyosin ATPase activity were reversed by treatment with 10 mM DL-dithiothreitol, a thiol-reducing agent. We conclude that the NO donor SNP inhibits contractile function caused by reversible oxidation of contractile protein thiols.

The effect of the excitatory amino acid receptor antagonist dizocilipine maleate (MK-801) on hemispheric cerebral blood flow and metabolism in dogs: modification by prior complete cerebral ischemia.

The effect of the N-methyl-D-aspartate (NMDA) receptor antagonist dizociplipine maleate (MK-801) on cerebral blood flow (CBF), cerebral metabolic rate for oxygen (CMRO2), intracranial pressure and systemic variables was examined in 6 normal dogs (Group I). In 6 additional dogs (Group II), the effects of a prior 11 min episode of complete cerebral ischemia on the response to dizocilipine was studied. CBF was measured with a sagittal sinus outflow technique and CMRO2 was calculated as the product of CBF and the arterial to sagittal sinus O2 content difference. Dizocilipine was administered as a 150 micrograms/kg i.v. bolus followed by a 75 micrograms.kg-1.h-1 infusion for 90 min. Plasma dizocilipine levels were greater than 25 ng/ml for the duration of the infusion. The CSF levels were approximately half the plasma levels. Five minutes after initiation of dizocilipine treatment, Group I dogs experienced a 63% increase in heart rate (P less than 0.01) and an 8% decrease in the mean arterial blood pressure (P less than 0.05). Over the same time interval. CBF increased by 85% (P less than 0.01) and intracranial pressure nearly doubled (P less than 0.05). In addition, dizocilipine treatment in all Group I animals resulted in EEG quasiperiodic bursts of delta-waves and polyspikes on a background of beta-activity. With the exception of the intracranial pressure, the above changes in systemic and cerebral variables persisted for the duration of the drug infusion. Intracranial pressure was no longer significantly elevated after 80 min of drug infusion. Hemispheric CMRO2 was unchanged by dizocilipine in Group I dogs. There was a decrease in the cortical glucose level at the end of the study, but no significant change in phosphocreatine, ATP, lactate, or energy charge when compared with 6 laboratory normals. An identical dose of dizocilipine administered after an 11 min episode of complete cerebral ischemia resulted in no significant changes in either cerebral or systemic variables. The absence of systemic effects in Group II dogs suggests that dizocilipine administration in normal dogs results in a centrally mediated activation of the peripheral sympathetic nervous system. The uncoupling of CBF and CMRO2 observed following dizocilipine treatment is similar to that reported for two other known NMDA antagonists, ketamine and phencyclidine. If administration of dizocilipine results in improved histopathological and neurological outcome following an episode of complete cerebral ischemia, this improvement is unrelated to changes in postischemic CBF or hemispheric CMRO2.

Evaluation of the glutamate antagonist dizocilipine maleate (MK-801) on neurologic outcome in a canine model of complete cerebral ischemia: correlation with hippocampal histopathology.

This study was designed to determine if dizocilipine maleate (MK-801), administered following 11 min of complete ischemia in dogs, could favorably alter neurologic outcome and hippocampal damage. Eighteen dogs were anesthetized and subjected to complete cerebral ischemia by temporary occlusion of the ascending aorta and the venae cavae via a thoracotomy. Five min postischemia, 9 dogs were given dizocilipine 150 micrograms/kg, followed by an infusion of 1.25 microgram/kg/min for 8 h. Control dogs were given equal volumes of placebo. Dogs were evaluated neurologically at 24, 48, and 72 h; thereafter, the brains were perfused, fixed and harvested. There was no significant difference in outcome between dizocilipine- and placebo-treated dogs: 5 of 9 given dizocilipine were normal, 1 was mildly injured and 3 were severely injured or dead. In the control animals given placebo, 3 of 9 were normal, 2 were mildly injured and 4 were moderately to severely injured. Histopathologic examination was limited to the hippocampus. CA1 and CA2,3,4 pyramidal neurons were graded according to degree of injury on a 5-point scale. There were no differences in histopathologic grades between the two groups. However, in both groups combined there was a significant correlation between neurologic outcome grade and histopathologic grade. The only notable systemic effect of dizocilipine appeared to be prolonged sedation which extended beyond 24 h postischemia but was not evident at 48 h postischemia. The authors conclude that more outcome studies in more sensitive models are needed.

Remote cerebellar hemorrhage after supratentorial surgery.

OBJECTIVE: Remote cerebellar hemorrhage (RCH) is an infrequent and poorly understood complication of supratentorial neurosurgical procedures. We retrospectively compared 42 patients who experienced RCH with a case-matched control cohort, to delineate risk factors associated with the occurrence of this complication. METHODS: Between 1988 and 2000, 42 patients experienced RCH after supratentorial neurosurgical procedures at our institution. Diagnoses were made on the basis of postoperative computed tomographic or magnetic resonance imaging findings in all cases. The medical records for these patients were reviewed and compared with those for a control cohort of 43 patients, matched for age, sex, surgical lesion, and type of craniotomy, who were treated during the same period. RESULTS: RCH most commonly occurred after frontotemporal craniotomies for unruptured aneurysm repair or temporal lobectomy and was frequently an incidental finding on postoperative computed tomographic scans. However, some cases of RCH were associated with significant morbidity, and two patients died. Preoperative aspirin use and elevated intraoperative systolic blood pressure were significantly associated with RCH (P = 0.026 and P = 0.036, respectively). Pathological findings for two cases demonstrated hemorrhagic infarctions in both. CONCLUSION: RCH most commonly follows supratentorial neurosurgical procedures, performed with the patient in the supine position, that involve opening of cerebrospinal fluid cisterns or the ventricular system (such as unruptured aneurysm repair or temporal lobectomy). Preoperative aspirin use and moderately elevated intraoperative systolic blood pressure are potentially modifiable risk factors associated with the development of RCH. Although RCH can cause death or major morbidity, most cases are asymptomatic or exhibit a benign course. Cerebellar "sag" as a result of cerebrospinal fluid hypovolemia, causing transient occlusion of superior bridging veins within the posterior fossa and consequent hemorrhagic venous infarction, is the most likely pathophysiological cause of RCH.

Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry.

Procedures are described for the treatment of phase and modulation lifetime data in fluorescent systems having multiexponential decay. All computer procedures (called FIT programs) arise from the lifetime resolution theory for phase-modulation measurements (Weber, G. (1981) J. Phys. Chem. 85, 949-953). The programs most successful in resolving heterogeneous lifetimes use a Monte Carlo approach in which phase and modulation lifetime data at three modulation frequencies are simultaneously utilized. These programs are shown to have more utility than the final closed form procedure presented by Weber (1981). The FIT routines are simple and require little computer time while yielding excellent results. To illustrate the applicability of these programs, defined binary (carbazole and pyrene) and ternary systems (carbazole, pyrene and POPOP) were examined. In most cases, the resolved lifetimes were within 5% of the independently measured value and the fractional fluorescence contributions were within 10% of that expected. These results demonstrate that phase-modulation measurements analyzed by appropriate computer programs are capable of solving for lifetimes in both binary and, in selected cases, ternary systems. An example is given from the recent literature (Dalbey, R., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696-4706) in which the above programs allowed the resolution of both binary and ternary lifetimes of a dansyl label on myosin, where Förster energy transfer was occurring. These lifetimes were used to quantify changes in distances between two activity-related thiols on myosin upon the addition of Mg-ATP or its analogs.

Three-dimensional computer modelling system for the study of biological structures.

A three-dimensional computer modelling system has been developed for use in biology, and is currently running on a Sun3 computer. The data originate as a series of two-dimensional micrographs which are digitised via a TV camera. The two-dimensional images are used to select features of interest and to construct a three-dimensional model. This model can be viewed in vector or solid format, it can be rotated about three orthogonal axes and can be viewed in three dimensions as a stereo pair or an anaglyph. The system has been used in a large number of projects over the past 10-15 years, for example, to examine physiological and nerve structures. The time-consuming part of the process is the selection of features, which involves a high level of biological expertise. Present developments are concerned with reduction of the time spent in feature recognition and involve the introduction of expert systems together with human-computer interaction to deal with problems of identification.

Automatic image analysis of antibody assays to measles virus.

A system for the automatic analysis of 24-well plates used in antibody assays to measles virus has been designed and developed based on digitising the information on the plate through a CCD camera, displaying the image and then analysing it using image processing methods. The system is being used in the analysis of sera from individuals vaccinated against measles and has been compared with the previous method where the plates were assessed by eye. The results from both methods are very similar although the manual method consisted of counting numbers of plaques (clear areas in the cells of the plate) and the automatic method measured plaque area. The automatic method is much faster than the original method and prevents operator fatigue. It does not deal, at present, with anomalies such as partially filled wells but could be developed to do so by incorporating intelligence into the system.

Control units for operation of computers by severely physically handicapped persons.

For those severely physically disabled persons who are unable to use keyboards, the computer has to be adapted to operate from the limited signals that can be obtained from controlled movements. These movements need to be identified and appropriate devices provided which can produce suitable signals to operate a computer. To minimize programming effort, it is also necessary to standardize on a particular form of input signal-in our case, one or two on/off contacts-for which the programs are developed. It is desirable for user input devices to be linked to the computer through an Interface Control Unit, which can also be operated from a remote infra-red unit mounted on a wheelchair. Such units are described.

Three-dimensional reconstruction of biological sections.

To observe internal detail in biological structures using light or electron microscopy, specimens need to be prepared from thin sections. Quantitative analysis of these section requires the transfer of complete section images, or selected features from them, into a computer. Where a complete structure is represented by a series of consecutive sections, images may be combined to represent the three dimensional structure. An interactive computer system is described which enables selected features of serial section images to be entered into the computer, edited, reconstructed in three-dimensions and displayed in any orientation. Sections can be analysed individually using appropriate, compatible programs, and an assessment of the complete structure obtained by interpolating between them. Categorisation of the different substructures within the sections allows them to be analysed and displayed separately.

Human-computer interaction and expert systems for three-dimensional studies of biomedical images.

Three-dimensional reconstruction of serial sections, observed by microscopy, is an important technique in medicine and biology. To view any part of a structure clearly, that part has to be identified and clearly highlighted in its relationship to other features in a structure. The identification process can be time consuming and tedious if many sections are involved, especially for routine applications, since human identification is required. In this paper we describe how image processing, together with other information on the shape and position of features relative to each other on any one section and throughout the structure, could be incorporated into an expert system and we also show how such a system could be designed. An important feature is the use of human-computer interaction to allow the system to evolve under the guidance of the biological or medical expert. An example of feature identification in a plant-parasitic nematode is used.