Procedure for the embedment and ultrastructural visualization of cells cultured on plastic microtest plates.

The polystyrene Microtest plate has served as an excellent means of quantitation in the interaction of immunological, chemotherapeutic and radiobiological treatments with cultured cells. In order to assess the accompanying ultrastructural changes it was necessary to develop a technique which allowed cells grown in the wells of the plates to be embedded for electron microscopy. Conventional epoxy resin embedding techniques require the use of propylene oxide as a clearing agent. Unfortunately propylene oxide is a solvent of the polystyrene plates. By the substitution of absolute ethanol for propylene oxide, toluidine blue staining during the procedure and other preparative techniques it was feasible to prepare reacted cells grown in Microtest plates for electron microscopy.

Microcystic variant of meningioma: a light-microscopic and ultrastructural study.

Meningiomas occasionally show small areas in which the cells are stellate with slender processes surrounding microscopic spaces, giving a lace-like appearance to the tissue. In the case of a 52-year-old woman with a tumor rising from the falx, virtually the entire tumor was composed of lacy reticulated tissue. Most of the tumor cells had processes that stained blue with phosphotungstic acid hematoxylin, suggesting the appearance of a glioma. However, electron microscopy showed that the extracellular space was extensive, and the tumor cells had long cytoplasmic processes which formed interdigitations with one another. Desmosomes were numerous. The immunoperoxidase method for glial fibrillary acidic protein gave negative results. On the basis of these histologic features, we suggest that this tumor was a microcystic variant of meningioma.

Immunoelectron microscopic study of substance P-containing fibers in feline cerebral arteries.

The ultrastructure of substance P-containing fibers in feline cerebral arteries was examined by combining substance P immunohistochemistry and electron microscopy. At the light and electron microscopic level, positive fibers were observed in the adventitia and at the border between the adventitia and media, but not within the media or the endothelium. The substance P-containing fibers were unmyelinated with diameters consistent with C-fiber caliber. Positive axons were in close contact with Schwann cell processes. Positive axons contained 24 nm microtubules, 10 nm neurofilaments, clear vesicles and scattered mitochondria. The number of mitochondria and organelles resembling vesicles appeared to increase in presumptive axon terminals. No synaptic membrane specializations were observed.

High-flow angiopathy: cerebral blood vessel changes in experimental chronic arteriovenous fistula.

Profound vascular damage secondary to high-flow extracranial states has been well characterized. However, changes in cerebral vasculature secondary to high-flow states have not been studied. To determine changes related to high-flow states in cerebral vessels, a rabbit model was developed in which torrential flow was created in the vertebrals, carotids, basilar, and vessels of the circle of Willis by means of a carotid-jugular shunt after ligation of the proximal carotid. The clinical, angiographic, and histologic changes noted in the animal model include: abrupt clinical deterioration after a variable interval with some animals developing ptosis, afferent vessel dilatation and the development of prominent anastomotic channels, variable cerebral vessel histopathology--related to duration and relative proximity to the shunt--affecting all three vessel layers, plump, irregular, and clumped endothelium, denuded with adherent platelets, irregular, duplicated, and thinned internal elastic membrane, frayed with invasion of the intima by mesenchymal cells, vacuolization and necrosis of the media muscle, and invasion of adventitia by foreign cells and small blood vessels. The high-flow angiopathy seen in this model may help explain vascular changes associated with high-flow cerebral vascular lesions, as well as other types of vascular damage.

Delayed CSF lavage for arteriographic and morphological vasospasm after experimental SAH.

Irrigation of the subarachnoid space after aneurysmal subarachnoid hemorrhage (SAH) has been reported to alleviate subsequent arterial vasospasm. The authors have investigated the effect of lavage of the cerebrospinal fluid (CSF) space in the two-hemorrhage canine model of vasospasm. Twelve dogs had basilar cistern lavage with 120 cc of artificial CSF 24 hours after each of two SAH's, and 12 control dogs had two sequential SAH's without intervening lavage of clot. The amount of clot on the ventral brain stem was evaluated at sacrifice and was graded from 0 (no clot) to 4 (maximum clot) to assess the adequacy of clot removal. Dogs that had undergone lavage had a median grade of 1 (range Grade 0 to 2); control dogs had a median grade of 2 (range Grade 1 to 3.5, p less than 0.001. Wilcoxon rank sum test), indicating significant reduction of gross clot by lavage. The neurological findings were graded from 0 to 5, based on meningismus, ataxia, paresis, and cranial nerve deficits. No significant differences in neurological grade were found on any day between the two groups. Satisfactory angiograms were obtained before and 7 days after hemorrhage and were controlled for blood pressure and blood gases; these showed significant spasm in both groups. There was a mean reduction (+/- standard deviation) of 21.6% +/- 16.2% in basilar artery diameter in control dogs, compared to a 28.8% +/- 15.1% reduction in dogs with lavage (difference not significant, t-test). There was a strong, but insignificant, trend toward reduction of endothelial desquamation in the basilar and middle cerebral arteries in dogs with lavage compared to control animals (p = 0.06). Corrugation and tearing of the elastica, thickened intima, intimal fibroplasia, vacuolization of the endothelial or smooth-muscle cells, and presence of blood cells in the adventitia occurred similarly in both groups. It appears that cisternal lavage 24 hours after hemorrhage in this model has no effect on the angiographic, neurological, or most morphological sequelae of SAH, in spite of evidence for removal of clot as seen at sacrifice. Any postulated interaction of clot and vessel resulting in chronic vasospasm must occur before this time. Evaluation of the effect of much earlier lavage (for instance, 1 hour after hemorrhage) may elucidate the point at which vasospasm is instigated after SAH, and help in determining what factors cause vasospasm.

Characteristics of human optic gliomas in tissue culture.

Nine human optic gliomas were examined in tissue culture. Typically, growth from the explants revealed well differentiated bipolar cells with abundant 9 to 10 nm fibers similar to those observed in the surgical specimens. Multinucleation was rare except for one culture, which had as many as 20 nuclei arranged in a palisading fashion along the periphery of some of the cells. Degenerative changes of the 9 to 10 nm fiber bundles with the production of amorphous electron-dense deposits were observed both in vivo and in vitro, and were thought to represent the formation of Rosenthal fibers. A distinctive feature of some of the optic gliomas was the ability of their long, thin cellular process to form fibrous tangles in tissue culture. The correlation of these fibrous tangles in culture with Rosenthal fibers in vivo is still uncertain.

Morphological changes of the basilar artery, ventricles, and choroid plexus after experimental SAH.

Experimental subarachnoid hemorrhage (SAH) was induced in adult rabbits by three repeated cisterna magna injections of autologous nonheparinized blood. One week after the last blood injection the animals were sacrificed and examined for morphological change. No vasculopathy was noted in the basilar arteries and no periarterial thrombus was seen around the majority of these arteries. Gross and microscopic changes were observed in both the ventricles and choroid plexus. Ventricular changes included dilation of the lateral ventricles, destruction of ciliated ependymal cells, and deposition of small amounts of blood throughout the ventricular system. These ventricular changes are similar to the pathological sequelae of SAH in patients. Choroid plexus changes included electron-dense cytoplasmic inclusions and dilation of the lateral and subcellular spaces. The ventricular surface and the choroid plexus are both affected by the intracisternal injection of blood. The hydrocephalus that follows SAH may be potentiated by ependymal disruption and loss of ciliated activity in the ventricles. Changes in choroid plexus function may also occur.

Cerebral arterial constriction after experimental subarachnoid hemorrhage is associated with blood components within the arterial wall.

Sequential cisternal blood injections in dogs reproduce some of the morphological and physiological features seen in man after subarachnoid hemorrhage-induced vasospasm. This study reports the morphological features observed in cerebral vessels in areas exposed to subarachnoid blood. Subarachnoid hemorrhage was produced in dogs by two cisternal injections of non-heparinized autologous blood 48 hours apart. Dogs were sacrificed 48 hours after the second injection. Angiographic narrowing of the basilar artery was routinely present 48 hours after the second injection, and there was a good correlation between angiographic vasospasm and a narrowed arterial lumen at postmortem examination. All basilar arteries showed structural changes with electron microscopic examination; these included endothelial cell vacuoles, early smooth-muscle cell necrosis, intimal changes, and adventitial erythrocytes, leukocytes, and mast cells. The finding that accompanied vessel constriction most uniformly was packing of the adventitial cerebrospinal fluid spaces with erythrocytes. Angiographically visible spasm was resistant to vasodilators. These data suggest that infiltration of blood elements into the arterial wall is an important concomitant feature of morphological and angiographic vasospasm.

Cerebrospinal fluid may nourish cerebral vessels through pathways in the adventitia that may be analogous to systemic vasa vasorum.

Cerebral blood vessels are devoid of vasa vasorum. Therefore, the authors have studied the microarchitecture of the adventitia of large feline cerebral vessels of the same size, in an effort of determine how the vessels are nourished. The cerebral vessels contain a rete vasorum in the adventitia that is permeable to large proteins and is in continuity with the subarachnoid space. This substructure may be analogous to the systemic vasa vasorum and may contribute to the nutrition of the cerebral arteries.

Isovolemic hemodilution in experimental focal cerebral ischemia. Part 2: Effects on regional cerebral blood flow and size of infarction.

Seventy-six splenectomized dogs were entered in a study of the value and effects of isovolemic hemodilution. Of these, seven were not included in the analysis because of technical errors. Of the remaining 69 dogs, 35 were treated with hemodilution; 28 were subjected to a 6-hour period of temporary occlusion of the distal internal carotid artery and the proximal middle cerebral artery, and seven underwent a sham operation only, with arterial manipulation but no occlusion. The other 34 dogs were not subjected to hemodilution; 26 of these underwent temporary arterial occlusion and eight had a sham operation only. In each group the animals were about equally divided into 1) an acute protocol with regional cerebral blood flow measurements by a radioactive microsphere technique and sacrifice at the end of the acute experiment, and 2) a chronic protocol with survival for 1 week to permit daily neurological assessment and final histopathological examination but without blood flow measurements. The general experimental protocol, the hemodynamic and rheological measurements, and the changes in intracranial pressure are described in Part 1 of this report. In the animals with arterial occlusion, blood flow decreased significantly in the territory of the ischemic middle cerebral artery. This decrease was partially reversed by hemodilution in the animals so treated. When the changes in blood flow before and after hemodilution in treated animals are compared with the changes at equivalent times in animals without hemodilution, the increases in flow in the gray matter of the ischemic hemisphere brought about by hemodilution are statistically significant. The neurological condition of the animals in the chronic protocol (sacrificed 1 week after occlusion) with hemodilution, as evaluated by daily neurological assessment, was significantly better than that of the control animals. In the animals sacrificed acutely (8 hours after arterial occlusion), the volume of infarction as estimated by the tetrazolium chloride histochemical method was 7.36% of the total hemispheric volume in the control animals and 1.09% in the hemodiluted animals, showing a statistically significant difference (p less than 0.005). In the chronic animals these values were 9.84% and 1.26%, respectively (p less than 0.005), as calculated by fluorescein staining. By histopathological examination the volume of infarction in the chronic animals was calculated as 10.92% in the control animals and 1.20% in the hemodiluted animals (p less than 0.005).(ABSTRACT TRUNCATED AT 400 WORDS)

Delayed cerebral vasospasm is not reversible by aminophylline, nifedipine, or papaverine in a "two-hemorrhage" canine model.

Angiographic spasm of cerebral arteries was produced in dogs by successive injections of cisternal blood 48 hours apart. Angiograms were taken before and after each cisternal injection. There was progressively greater angiographic vasospasm of the basilar artery. Intravenous aminophylline, 10 mg/kg/hr, reversed vessel constriction on the 1st and 3rd day after one injection of cisternal blood. On the 5th day after two blood injections (on Day 1 and Day 3), intravenous aminophylline, nifedipine (1 mg/kg), and intra-arterial bolus injection of 2 mg/kg papaverine failed to reverse the constriction. The intractable constriction produced in this model resembles that found in patients. The calcium antagonist, nifedipine, is as ineffective as the more traditional vasodilators in reversing vasospasm in this model.

Immunological, biochemical, ultrastructural, and electrophysiological characteristics of a human glioblastoma-derived cell culture line.

This report presents the results of a study using multiple techniques of the established human cell line, LM, which has been developed in culture medium from a patient with a right temporoparietal glioblastoma. This cell line has human subtetraploid karyotype and has several features of a transformed line in culture. These include continuous propagation for 10 years, ability to form tumor nodules when transplanted into immunologically suppressed hamsters, and pleomorphic appearance. Ultrastructurally, it is characterized by multiple nuclei, few actin cables, and numerous surface-membrane microvilli, as well as abundant 9- to 10-nm cytoplasmic filaments. By its immunological reactivity, the line can be shown to contain glial fibrillary acidic protein at low levels, consistent with its glial origin and continued nature. Dibutyryl cyclic adenosine monophosphate (db-cAMP) induces formation of long astrocytic-like processes as well. Its membrane electrical characteristics include a low resting membrane potential and short time constant. Used in a microtiter antiglioma antibody cytotoxicity assay, LM yields a positive reaction to antibodies in the sera of 80% of patients with astrocytomas and only 9% of normal blood-bank donors, suggesting that it shares common antigens with other astrocytic tumor lines. The varied characteristics of this glioblastoma-derived line emphasize the "multiforme" nature on the neoplasm and suggest that for characterization of any such line, multiple parameters are necessary to allow comparison with other long-term glioblastoma lines in the literature. The usefulness of the LM line in in vitro cell biological, immunological, chemotherapeutic, and radiobiological studies of gliomas makes such efforts very worthwhile.

Obliteration of experimental aneurysms in dogs with isobutyl-cyanoacrylate.

Experimental cervical carotid aneurysms in dogs are obliterated with isobutyl-cyanoacrylate ( IBCA ) injected under direct vision into the aneurysm. Reflux of IBCA into the artery was prevented by inflating either a latex or a Silastic balloon in the carotid artery at the level of the neck of the aneurysm. This balloon was introduced through a catheter advanced into the common carotid artery by femoral catheterization. The Silastic balloon was found to be much more effective than the latex balloon in preventing spillage of IBCA into the lumen.

Ultrastructural and electrophysiological features of meningioma whorls in tissue culture.

Cultured cells derived from a psammomatous meningioma were induced to from whorls in vitro by deprivation of nutrients. The ultrastructural features of desmosomes and junctional complexes were similar to sections from the surgical specimen; also prominent were 9-10 nm cytoplasmic filaments. Microelectrode recording of whorled cells showed a larger resting membrane potential than that of similar isolated meningioma cells in culture. Whorl formation seen in this tumor may be a result of relative nutritional deprivation and the need for metabolic cooperation among tumor cells.

Arginine vasopressin causes morphological changes suggestive of fluid transport in rat choroid plexus epithelium.

The experiments described herein use an in vitro preparation of choroid plexus to demonstrate that it is a vasopressin-responsive organ by morphologic criteria. Choroid plexus from rats was incubated for one hour in graded concentrations of arginine vasopressin (AVP). Within physiologic range of molar concentration, incubation in vasopressin induced a decrease in basal and lateral spaces in choroid plexus epithelial cells as well as an increase in number of dark cells. The number of cells with basal spaces decreased significantly from 82.7 +/- 9.2 in control tissue to 19 +/- 18 in tissue incubated in 10(-12) M AVP; similarly, the number with lateral cellular spaces decreased from 20 +/- 8.8 to 7.6 +/- 2.2 cells in 10(-10) M AVP. Dark cells increased in number from 3.8 +/- 2.6 in control conditions to 49 +/- 4 with 10(-9) M vasopressin. These data suggest important effects of arginine vasopressin in cerebrospinal fluid (CSF) on choroid plexus, compatible with enhanced fluid transport across choroid epithelial cells.

Ultrastructure of human endometrial epithelium in monolayer culture with and without steroid hormones.

Colonies of cells of epithelioid appearance were identified in monolayer cultures grown up to 50 days from normal human endometrial cell suspension obtained by a method designed to insure a maximum harvest of glandular cells. Groups of these cells were separated from stromal cells by means of cloning cylinders. Studies comparing the ultrastructure of cells of this type to fresh endometrial tissue revealed a number of similarities. The morphological characteristics common to both types of samples included junctional complexes, perinuclear microfilaments and microvilli with glycocalyx. Other common features were prominent nucleoli, well developed Golgi, rough endoplasmic reticulum and membrane-bound electron-dense bodies in the cytoplasm. A stripping technique applied to the fetal bovine serum used in the nutrient medium made it possible to initiate cultures in a steroid-free environment and to maintain them in the presence of the specified concentration of estradiol and/or progesterone. Isolation of epithelial cells of endometrium in monolayer culture may provide a useful model system in which to study the specific effects of steroid hormones on cellular function and differentiation.

Morphological, biochemical, ultrastructural, tissue culture and clinical observations of typical and aggressive craniopharyngiomas.

Craniopharyngiomas are tumors of the suprasellar area, which are often cystic, encapsulated and slow-growing. Certain of these tumors can behave in an aggressive manner and either invade surrounding structures or recur. In order to determine characteristics which may aid in distinguishing typical from atypical lesions, a study of biopsy and tissue culture specimens from 25 human craniopharyngiomas was undertaken. Tissue culture observations reveal two distinct cell populations. Typical lesions grew in culture in an orderly epithelial pattern and had desmosome-tonofibril aggregates and smooth surface topography demonstrable by electron microscopy. In the atypical tumors the cell growth was irregular, with mitotic activity, cholesterol crystals and features characteristic of neoplastic transformation, such as surface microvilli, an increase of cytoplasmic basophilia, size and number of nucleoli and retraction of cytoplasm. Correlation with the clinical status of the patients suggests that tumors of the four patients which exhibited atypical features in culture behaved more aggressively.

The microcirculation of cerebral arteries: a morphologic and morphometric examination of the major canine cerebral arteries.

A morphologic and morphometric examination of the major cerebral blood vessels in the dog was carried out to determine whether there were vasa vasorum in these arteries and what features might be associated with them. True vasa vasorum confined to the media were not seen in any of the vessels examined. Microvessels confined to the adventitia, however, were found in the internal carotid and vertebral arteries but not in the basilar, middle cerebral, or anterior spinal arteries. Animal size, vessel size as determined by adventitial and medial area, and the number of smooth muscle cell lamellae were not associated with the presence of these adventitial vessels; they occurred only in arteries with both an intra- and extradural portion. It therefore appears that most canine cerebral arteries do not have vasa vasorum.

Characterization and radiobiologic parameters of medulloblastoma in vitro.

Cultured cells from human medulloblastomas were demonstrated to have distinctive features and a "glial" potential from the morphological and ultrastructural parameters. The in vitro radiobiological survival curve parameters of cultured human medulloblastoma and non-malignant skin fibroblasts obtained under the same conditions of exponential growth were similar. In vitro conditions may underestimate the shoulder region of the survival curve. Since the shoulder region is the most important in clinical fractionated radiotherapy, multiple dose experiments might be useful in examining this portion of the survival curve more closely.