Biomedical subjects
Publications and source records attributed to R L Schultz.
Relating to the protocol by Mikki et al. [Brain Res. Protocols 2 (1997) 9-16], the use of an image analysis system is recommended in place of micrographs and photoprints for the counting and measuring of neuronal nuclei.
Previous methods for determining morphological features of vascular networks in cerebral cortex were subject to arbitrary variation and bias. Unbiased estimates of vessel number, volume, surface area and length can be obtained using stereology but these techniques tend to be tedious and time-consuming. Stereologic protocols generally require micrographs that have to be analyzed manually for intersections of vessels on grid points or lines. In this report, we provide a simpler and more precise method for measuring morphological features of cerebral cortical microvessels. Images of microvessels in 1 microm toluidine blue stained sections were captured using a popular image analysis software package. Luminal surfaces of endothelial cells were automatically traced using commonly available features; the two-dimensional data of vessels (diameter, area, perimeter and number of vessels) were automatically computed and transferred to a spreadsheet. Three-dimensional features were then determined using basic stereologic equations. The method eliminates the need for manual measurements and is particularly time- and cost-effective for quantitative studies where numerous images have to be evaluated.
Brain cells manufacture and secrete angiogenic peptides after focal cerebral ischemia, but the purpose of this angiogenic response is unknown. Because the maximum possible regional cerebral blood flow is determined by the quantity of microvessels in each unit volume, it is possible that angiogenic peptides are secreted to generate new collateral channels; other possibilities include neuroprotection, recovery/regeneration, and removal of necrotic debris. If the brain attempts to create new collaterals, microvessel density should increase significantly after ischemia. Conversely, if angiogenic-signaling molecules serve some other purpose, microvessel densities may increase slightly or not at all. To clarify, the authors measured microvessel densities with quantitative morphometry. Left middle cerebral arteries of adult male Sprague-Dawley rats were occluded with intraluminal nylon suture for 4 hours followed by 7, 14, 19, or 30 days of reperfusion. Controls received no surgery or suture occlusion. Changes in microvessel density and macrophage numbers were measured by light microscopic morphometry using semiautomated stereologic methods. Microvessel density increased only in the ischemic margin adjacent to areas of pannecrosis and was always associated with increased numbers of macrophages. Ischemic brain areas without macrophages displayed no vascularity changes compared with normal animals. These data suggest that ischemia-induced microvessels are formed to facilitate macrophage infiltration and removal of necrotic brain.
Numerous cytochemical studies have reported that calcium-activated adenosine triphosphatase (Ca2+-ATPase) is localized on the abluminal plasma membrane of mature brain endothelial cells. Since the effects of fixation and co-localization of ecto-ATPase have never been properly addressed, we investigated the influence of these parameters on Ca2+-ATPase localization in rat cerebral microvessel endothelium. Formaldehyde at 2% resulted in only abluminal staining while both luminal and abluminal surfaces were equally stained following 4% formaldehyde. Fixation with 2% formaldehyde plus 0.25% glutaraldehyde revealed more abluminal staining than luminal while 2% formaldehyde plus 0.5% glutaraldehyde produced vessels with staining similar to 4% and 2% formaldehyde plus 0.25% glutaraldehyde. The abluminal reaction appeared unaltered when ATP was replaced by GTP, CTP, UTP, ADP or when Ca2+ was replaced by Mg2+ or Mn2+ or p-chloromercuribenzoate included as inhibitor. But the luminal reaction was diminished. Contrary to previous reports, our results showed that Ca2+-specific ATPase is located more on the luminal surface while the abluminal reaction is primarily due to ecto-ATPase. The strong Ca2+-specific-ATPase luminal localization explains the stable Ca2+ gradient between blood and brain, and is not necessarily indicative of immature or pathological vessels as interpreted in the past.
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Cytochemical data in the literature reporting localization of sodium, potassium adenosine triphosphatase (Na(+), K(+)-ATPase) in the blood-brain barrier (BBB) have been contradictory. Whereas some studies showed the enzyme to be located exclusively on the abluminal endothelial plasma membrane, others demonstrated it on both the luminal and abluminal membranes. The influence of fixation on localization of the enzyme was not considered a critical factor, but our preliminary studies showed data to the contrary. We therefore quantitatively investigated the effect of commonly used fixatives on the localization pattern of the enzyme in adult rat cerebral microvessels. Fixation with 1%, 2%, and 4% formaldehyde allowed deposition of reaction product on both the luminal and abluminal plasma membranes. The luminal reaction was reduced with increasing concentration of formaldehyde. Glutaraldehyde at 0.1%, 0.25%, 0.5%, in combination with 2% formaldehyde, drastically inhibited the luminal reaction. The abluminal reaction was not significantly altered in all groups. These results show that luminal localization of BBB Na(+), K(+)-ATPase is strongly dependent on fixation. The lack of luminal localization, as reported in the literature, may have been the result of fixation. The currently accepted abluminal polarity of the enzyme should be viewed with caution.
It is presently believed that sodium, potassium-activated adenosine triphosphatase (Na+, K+-ATPase) is localized on the abluminal plasma membrane of brain endothelial cells. But there have been contrary reports from some cytochemical studies. We examined the localization of the enzyme in rat cerebral microvessel endothelium using the in situ model originally employed to establish the abluminal polarity concept. Alterations in fixation and incubation media from the original reports were conducted to determine the effect on localization pattern. With the Ernst indirect incubation method as originally used, three types of localization patterns were obtained: abluminal only, luminal only, and on both surfaces of endothelial cells. With the direct incubation method of Mayahara, reaction product was seen on both surfaces. Reduction in fixation time followed by the use of the indirect incubation method resulted in a complete loss of the reaction product. The same reduction in fixation time followed by the use of the direct method did not alter the localization pattern of the enzyme. Our results demonstrated that Na+, K+-ATPase is localized on both surfaces of brain endothelial cells. The localization pattern of Na+, K+-ATPase is significantly dependent upon fixation and the incubation medium used in the in situ model. Data discrepancies for the enzyme as reported in the literature appear to be caused by differences in cytochemical protocols, rather than the biological reasons advocated by other investigators. We conclude that past cytochemical reports of blood-brain barrier (BBB) Na+, K+-ATPase abluminal localization were incomplete. The currently held abluminal polarity theory of the enzyme needs to be reexamined. Past basic and clinical cytochemical studies of BBB Na+, K+-ATPase should be viewed and interpreted with caution.
Myelin/oligodendrocyte specific protein was compared to glial fibrillary acidic protein and 2'3'-cyclic nucleotide 3'-phosphodiesterase expression in normal rat brains and following stab wounds to the cerebral cortex, corpus callosum and hippocampus. Animals with stab wounds were allowed to recover for 5, 15, 28, 45 and 70 days post-operation before fixation by perfusion. Sections were reacted with antibodies against myelin/oligodendrocyte specific protein, glial fibrillary acidic protein and 2'3'-cyclic nucleotide 3'-phosphodiesterase, and observed by light and electron microscopy. Normal cerebral cortex had very few myelin/oligodendrocyte specific protein-positive and 2'3'-cyclic nucleotide 3'-phosphodiesterase-positive cells, but some glial fibrillary acidic protein-positive cells. The myelinated fibres of the corpus callosum were heavily stained for myelin/oligodendrocyte specific protein but unstained by glial fibrillary acidic protein or 2'3'-cyclic nucleotide 3'-phosphodiesterase antibodies. Some immunopositive cells were present in the corpus callosum and hippocampus with all three antibodies. After stab wound myelin/oligodendrocyte specific protein-positive reactive cells had more and longer processes and stained more intensely than equivalent cells in normal brain. These cells were distributed along the wound track, including within the cerebral cortex. The numbers of these cells increased until 28 days post-operation and then decreased so that very few were found at 70 days post-operation except in the corpus callosum. Where demyelination occurred myelin/oligodendrocyte specific protein-staining was lost. Staining for 2'3-cyclic nucleotide 3'-phosphodiesterase revealed a similar pattern. Glial fibrillary acidic protein-positive reactive cells, which were also more robust than the normal cells, were more widely distributed. They increased in number throughout the time periods studied and gliosis was evident on the contralateral side. The glial fibrillary acidic protein-positive astrocytes were also different from the myelin/oligodendrocyte specific protein-positive and 2'3'-cyclic nucleotide 3'-phosphodiesterase-positive oligodendrocytes in terms of cell shape. With electron microscopy myelin/oligodendrocyte specific protein-positive cells showed features typical of immature oligodendrocytes. We conclude that the injury caused a numerical increase in oligodendrocytes and that myelin/oligodendrocyte specific protein is a good marker for the oligodendroglial response and demyelination in pathological conditions.
We have identified very low density (VLDL) and low density (LDL) lipoproteins as blood plasma components that enhance the binding and deposition of fibronectin into the extracellular matrices of cultured MG-63 osteosarcoma cells and human fibroblasts. The lipoproteins increased the binding and deposition of iodinated fibronectin by MG-63 cells threefold over control levels. LDL also increased the deposition of multimeric fibronectin into extracellular matrix as assessed by gel electrophoresis and fluorescence microscopy. High density lipoprotein (HDL) and the d > 1.21 g/ml nonlipoprotein fraction had less activity. Enhancement of binding of fibronectin was observed within 15 minutes, when binding was largely reversible. LDL also increased the binding of a fragment containing the 70-kd amino-terminal region of fibronectin that is primarily responsible for the reversible binding of fibronectin to cell layers. LDL had to be present simultaneously with radiolabeled fibronectin to exert an effect on fibronectin binding. LDL enhanced fibronectin binding equally well to normal skin fibroblasts and to familial hypercholesterolemic fibroblasts lacking the LDL receptor. Acetylation of LDL, performed to block its interaction with the LDL receptor, did not diminish the enhancement of fibronectin binding to MG-63 cells. These results indicate that LDL and VLDL interact with fibronectin to potentiate binding to monolayer cells through a pathway that does not involve the LDL receptor.
Recent investigations have proposed that, after temporary ischemia, pentastarch may reduce microvascular permeability and reperfusion injury. However, this hypothesis has not been tested in the brain. Accordingly, after 180 min of temporary middle cerebral artery occlusion, the effect of pentastarch or albumin on blood-brain barrier permeability and cerebral injury was investigated in isoflurane-anesthetized rats. One of the following was maintained for the final 60 min of occlusion and throughout reperfusion: control-hematocrit was not manipulated; pentastarch-hematocrit was decreased to approximately 30% with pentastarch; or albumin-hematocrit was decreased (approximately 30%) with albumin. Part A (n = 21): 30 min of reperfusion was allowed, and blood-brain barrier permeability was determined with the indicator dye Evans Blue. Part B (n = 14): in different animals, 120 min of reperfusion was allowed, and cerebral injury (2,3,5-triphenyltetrazolium chloride stain) and edema (specific gravity) were assessed. Part C (n = 4): in different animals, the blood-brain barrier was evaluated by electron microscopy. Evans Blue (micrograms per gram brain tissue, mean +/- SD) was greater in the control (20.8 +/- 9.0) and albumin (15.5 +/- 7.3) groups versus the pentastarch (4.7 +/- 2.7) group (P less than 0.05). Brain injury (percent of hemisphere ipsilateral to occlusion) was less and specific gravity greater in the pentastarch (33 +/- 8 and 1.040 +/- 0.003 respectively) versus the albumin group (45 +/- 6 and 1.035 +/- 0.003). This study supports the hypothesis that during temporary cerebral ischemia, pentastarch decreases brain injury and edema.(ABSTRACT TRUNCATED AT 250 WORDS)
After 180 min of temporary middle cerebral artery occlusion in rats, the affect of phenylephrine-induced hypertension on blood-brain barrier permeability was assessed. One of the following blood-pressure regimens was maintained during either a 30- or 120-min period of reperfusion: (a) 30/Norm, 30 min of normotensive reperfusion was allowed; (b) 30/HTN, mean arterial blood pressure was increased by 35 mm Hg during 30 min of reperfusion; (c) 120/Norm, 120 min of normotensive reperfusion was allowed; or (d) 120/HTN, mean arterial blood pressure was increased by 35 mm Hg during 120 min of reperfusion. Evans blue (30 mg/kg) was given, and brains were analyzed for Evans blue by spectrophotometry. Evans blue (microgram/g brain tissue, mean +/- SD) was greater (P less than 0.05) in both hypertensive groups versus their time matched normotensive groups (30/HTN: 80 +/- 16 versus 18 +/- 6 in the 30/Norm group; 120/HTN: 17 +/- 6 versus 8 +/- 3 in the 120/Norm group). In addition, Evans blue was greater (P less than 0.05) in both 30-min groups versus their pressure matched 120-min groups (30/Norm: 18 +/- 6 versus 8 +/- 3 in the 120/Norm group; 30/HTN: 80 +/- 16 versus 17 +/- 6 in the 120/HTN group). The data are consistent with previous studies which have demonstrated an opening of the blood-brain barrier at the onset of reperfusion. In addition, the data support a hypothesis that changes in blood-brain barrier permeability are more sensitive to hypertension in the early period of reperfusion.
Pineal "synaptic" ribbons are a heterogeneous population of organelles. "Synaptic" ribbons (SR) sensu stricto, "synaptic" spherules (SS), and intermediate forms (IMF) are present. Their function and origin are unknown, and a knowledge of their prenatal development is lacking. Thus the pineal glands of prenatal, neonatal, and adult guinea pigs were prepared for electron microscopy. "Synaptic" ribbons were studied morphologically and quantitatively. The three categories of "synaptic" ribbons reported in adult pineal glands were also present in prenatal pineal glands. Their structural features, distribution, grouping, and composition patterns are similar to those in adults. "Synaptic" ribbons were first detected in pinealocytes of the distal region of a 42-day postcoitus (PC) pineal gland and were comparable with those in adults. They increased in number with age and reached a peak at 63 days PC, followed by a steep decline at 66 and 67 days PC. By day 69 PC, the numbers increased again and showed a dramatic increase after birth. Several true ribbon synapses were seen at day 63 PC between pinealocyte cell processes or between pinealocyte cell process and pinealocyte cell body. Since true ribbon synapses have not been found in adult guinea pig pinealocytes, their synaptic nature could have been lost during development. No precursors for the "synaptic" ribbons were found. The endoplasmic reticulum cisternae may be the origin for the ribbon vesicles because of their close association with the "synaptic" ribbons.
The ultrastructural localization of acetylcholinesterase (AChE) activity in guinea pig pineal gland was studied using the copper-glycine procedure. A small number of pinealocytes and bundles of unmyelinated nerve fibers were labeled by the AChE reaction. The AChE-positive pinealocytes were located near blood vessels and distributed in small groups. The AChE reaction product was localized in the perinuclear cistern, in the cisternae of the endoplasmic reticulum (ER), and in the saccules of the Golgi apparatus. These findings suggest that the AChE-positive pinealocytes synthesize AChE. The AChE reaction product was also seen in the intercellular space between pinealocyte processes. Besides pinealocytes, AChE activity was localized on the axolemma of myelinated and unmyelinated nerve fibers and in the basement membrane surrounding unmyelinated nerve fibers. Pseudocholinesterase activity was confined to Schwann cells, which showed the reaction product in their perinuclear cistern, in the cisternae of the ER, and on the plasmalemma.
Thiamine pyrophosphatase (TPPase), nucleoside diphosphatase (NDPase), and glucose-6-phosphatase (G-6-Pase) were localized by the cerium technique in guinea pig pinealocytes and compared with the corresponding lead technique. NDPase and TPPase were also compared at different pH values using the cerium technique. Vibratome sections of perfusion-fixed tissue were incubated with cerium chloride or lead nitrate. Substrates used were thiamine pyrophosphate (for TPPase), sodium inosine diphosphate (NDPase), and disodium glucose-6-phosphate (G-6-Pase). The 1-2 trans saccules of the Golgi apparatus showed TPPase and NDPase activity but none for G-6-Pase. The endoplasmic reticulum (ER) cisternae and perinuclear space had NDPase and G-6-Pase activity but not TPPase. The abluminal plasmalemma of endothelial cells and the plasmalemma of Schwann cells demonstrated TPPase and NDPase activity but the luminal plasmalemma of the endothelial cells and the plasmalemma of pinealocyte processes showed only NDPase activity. TPPase was active at all pH values tested, but NDPase was most active at pH values of 6.5 and 7.0. Lead phosphate precipitate was frequently seen in nuclei, perinuclear space, ER cisternae, and "synaptic" vesicles when lead was used as the capturing agent. These sites were usually not labeled when cerium was used.
The relationships of tibial endosteal osteoclasts to bone surfaces were quantitatively evaluated during initiation of calcium repletion in calcium-deficient rats. To do this, indices of osteoclast-bone relationships obtained by light microscopy were devised and evaluated by comparing with those obtained by electron microscopy (EM). These indices are the percent of the osteoclast width that (1) exhibits markers indicative of a ruffled border, (2) is in close contact with bone, (3) is isolated from bone by other cell types, and (4) is separated from bone by intercellular material. The indices obtained by light microscopy were strongly correlated with similar indices obtained by EM and were equally sensitive but considerably easier to obtain. The ruffled border and contact index were significantly decreased by 3 hours after beginning the meal whereas cells of other types became interposed between the osteoclasts and the bone.
The common procedures used for preparing some organs and tissues for electron microscopy, in which a fixative with the buffer portion adjusted to near-isotonicity to plasma is perfused in vivo, causes intolerable shrinkage of rat pineal cells. The present study was undertaken to optimize the parameters involved in the fixation of the pineal gland. The buffer and its concentration and the aldehyde or aldehydes used were among the variables investigated. The buffers tried were phosphate, cacodylate, PIPES, and HEPES. Decreasing the buffer concentration prevented shrinkage with all four buffers. The optimum concentrations were 0.05 M phosphate, 0.07 M cacodylate, 0.05 M or 0.057 M PIPES, and 0.1 M HEPES. PIPES and HEPES were clearly superior in retaining cytoplasmic density when compared with phosphate or cacodylate. The use of lithium PIPES and HEPES instead of the sodium equivalents enhanced membrane detail. A small volume of more concentrated aldehyde fixative perfused ahead of the main perfusate (a strong prewash) definitely helped prevent shrinkage. Using a mixture of aldehydes consisting of glutaraldehyde, formaldehyde, and acrolein reduced the tendency for shrinkage when compared with glutaraldehyde only. Some of the shrinkage space artefacts could be easily misinterpreted as normal features. Since the pineal gland commonly contains degenerating structures, a dependable fixation procedure is particularly needed. Also, accurate preservation is essential in the evaluation of physiological changes.
Vitronectin, also known as serum-spreading factor or S-protein, mediates cell adhesion and inhibits formation of the membrane-lytic complex of complement and the rapid inactivation of thrombin by antithrombin III in the presence of heparin. Vitronectin is normally present in plasma at a concentration of approximately 300 micrograms/mL. The investigators quantified plasma vitronectin with an enzyme-linked immunosorbent assay and visualized reduced and nonreduced vitronectin by immunoblotting after separation of plasma or serum by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of plasma vitronectin was markedly reduced in some patients with disseminated intravascular coagulation, especially in those with liver failure; it was near normal in patients with metastatic cancer and acute leukemia. Patients with vitronectin levels less than 40% normal invariably had low fibrinogen and antithrombin III and a prolonged prothrombin time. In both normal and patient plasmas there was heterogeneity in the ratio of the 75,000- and 65,000-mol wt polypeptides of reduced vitronectin: 18% had mostly the 75,000-mol wt polypeptide, 59% had roughly equal amounts of the two polypeptides, and 22% had mostly the 65,000-mol wt polypeptide. This polymorphism is inherited and appears to be due to two alleles that are present with approximately equal frequency. The blotting patterns of vitronectin in reduced and nonreduced plasmas were largely unaltered in plasma of patients with defibrination syndrome, fibrinolysis, liver failure, sepsis, metastatic cancer, and acute leukemia. There was no evidence of fragmentation of vitronectin or formation of the disulfide-bonded complex of vitronectin and thrombin-antithrombin III that is found when blood is clotted. Thus these results corroborate in vitro observations that the liver is the major source of plasma vitronectin, suggest that vitronectin may become depleted during disseminated intravascular coagulation, and define a genetic polymorphism of vitronectin.
When used for vascular perfusion of brain, 0.1 M PIPES-buffered 3% glutaraldehyde resulted in the formation of expanded, vesicle-filled cell processes limited by multiple membrane layers. These structures, termed multivesicular myelin figures and interpreted as artefacts, were most common in layer 2 of the cerebral cortex. When cacodylate or phosphate buffer was used instead of PIPES buffer in the primary fixative, such structures were not seen. The use of a more concentrated initial aldehyde fixative, PIPES-buffered, markedly reduced the size and numbers of these artefacts when compared to PIPES-buffered 3% glutaraldehyde only. Slowing the initial perfusion rate increased the size and frequency of occurrence of multivesicular myelin figures with PIPES buffer when compared to optimum perfusions. Prolonged initial exposure to PIPES buffer by using it to wash out the blood and then perfusing with fixative 5 min later did not increase the number or size of multivesicular myelin figures but did reduce the multivesicular nature of the artefacts. We suggest that the non-toxic nature of PIPES buffer allowed the formation of these membranous artefacts, while phosphate and cacodylate interfered with the cellular activity during the process of fixation.