A major problem facing ischemic stroke therapy is the lack of treatments that directly restore the anatomy and physiology of the injured neurovascular unit. Therapeutic angiogenesis emerged as a potential treatment for ischemic stroke after preclinical studies demonstrated that neovascularization induced by angiogenic pharmacological agents is associated with neuroprotection. This chapter discusses the: (1) epidemiology of ischemic stroke and the limitations of current treatments, (2) neuroprotection and therapeutic angiogenesis as new treatments for ischemic stroke, (3) biology of vascular endothelial cell growth factor (VEGF) and how VEGF has become a prime candidate for therapeutic angiogenesis, (4) potential clinical benefits and adverse effects of VEGF-based therapeutic angiogenesis for ischemic stroke, and (5) gaps in knowledge requiring further preclinical investigations before VEGF-based therapeutic angiogenesis can be considered safe and effective to begin clinical trials for ischemic stroke patients. The unresolved issues in the preclinical trials are whether: (A) VEGF-based therapeutic angiogenesis promotes or hinders neuroprotection, (B) doses of VEGF not demonstrating adverse effects at the light microscopy level associated with clinically-significant ultrastructural alterations of the neurovascular unit, (C) VEGF combination therapy provide greater neuroprotection over VEGF monotherapy without additional adverse effects, (D) different isoforms of VEGF produce different therapeutic outcomes, and how the most beneficial isoform affects the anatomy and physiology of other organs, (E) VEGF-based therapeutic angiogenesis affects systemic hemodynamics, (F) different animal models of ischemic stroke produce similar favorable or adverse outcomes, including the influences of age, gender and coexisting chronic diseases, (G) gene therapy and stem cells are beneficial for VEGF-based therapeutic angiogenesis for stroke.

Therapeutic angiogenesis by vascular endothelial growth factor (VEGF) is advocated as a promising treatment strategy for brain ischemic stroke. However, data in the literature demonstrating the benefit of therapeutic angiogenesis are contradictory. In this paper, we describe the effects of non-angiogenic and angiogenic doses of VEGF165 on macrophage density and histology of normal and ischemic brains of adult rats. VEGF165 was administered intra-arterially for 7 days following temporary occlusion of the middle cerebral artery. In contrast to ischemic brains treated with non-angiogenic doses of VEGF165 which showed preserved neuropil and reduced numbers of macrophages, ischemic brains treated by an angiogenic dose showed phagocytized neuropil and high macrophage density. Though neither non-angiogenic nor angiogenic doses caused macrophage infiltration in normal brains, damage of the brain matrix occurred with the angiogenic dose. These results suggest an angiogenic dose of VEGF165 injures the nervous tissue rather than promote recovery. Angiogenesis by VEGF monotherapy for ischemic stroke should be viewed with caution, or avoided. Since our data show intravascular administration of VEGF165 does not cause macrophage inflammation, in contrast to reports in the literature whereby VEGF165 was applied directly to the brain, our findings also indicate the relationships between VEGF, angiogenesis, and macrophage inflammation are governed by the route VEGF is administered to the brain.

There are different interpretations of tethered cord syndrome (TCS) partly due to difficulty in understanding the concept of this syndrome as a functional disorder not merely based on gross anatomy of congenital anomalies. The essential mechanical factor of cord tethering is that any of the inelastic structures fastening the caudal end of the spinal cord produces traction effects on the lumbosacral cord. The production of such traction is the key to understanding this disorder. In a significant number of patients who present with the typical clinical signs and symptoms of TCS, the diameter of the filum terminale is found within normal limits and the caudal end of the spinal cord is located in the normal position. Therefore, the definition of TCS requires the demonstration that there is a posterior displacement of the conus and filum by MRI, lack of viscoelasticity by the stretch test of the filum during surgery, and fibrous displacement of glial tissue within the filum by histological studies. This is because there is inconsistency from such studies as ultrasonography, MRI and CT myelography, which attempt to establish the presence of a tight filum terminale. A goal of this article is to provide basic understanding of TCS so that clinicians can use the concept of stretch-induced spinal cord dysfunction for proper diagnosis and treatment of this disorder.

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.

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.

For the cytochemical demonstration of enzymes, the commonly employed procedures involve using chopped small cubes or sections of tissue incubated in a proper medium. With the chopped tissue, penetration of substrate and capturing agents is very slow. When using frozen sections artifacts are difficult to avoid. Also proper vibratome sections are hard to achieve with small organs such as the pineal gland. In order to overcome these disadvantages, we have developed a vascular perfusion incubation technique for the demonstration of AChE.With this technique, a simple gravity flow apparatus for vascular perfusion with two glass containers for fixative and Tyrode solution was used. A needle (19g) with a 32 cm long vinyl tube was connected to the apparatus by a three-way valve. Adult Djungarian hamsters were anesthetized with 1.5 g/kg urethane. The needle was inserted into the ascending aorta through the left ventricle. The following solutions were then perfused for the times indicated: Tyrode, 1 min; fixative (1% purified glutaraldehyde and 2% formaldehyde, 0.05 M cacodylate buffer, pH 7.2), 5 min; Tyrode, 10 min; substrate-free incubation medium, 10 min; complete incubation medium, 20 min; pause for 10 min; repeat of the last two steps four times; isotonic sodium sulphate, 10 min; buffered sulphide, 30 min; isotonic sodium sulphate, 10 min; fixative (3% glutaraldehyde and 2% formaldehyde, 0.05 M cacodylate buffer), 5 min. The entire pineal gland and pieces of the tongue were taken. After osmium postfixation standard electron microscopic procedures were used.

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 (μg/g brain tissue, mean ± SD) was greater (P<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<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.

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.

When used for vascular perfusion of brain, 0.1m 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.

Insulated, bipolar stainless steel electrodes were chronically implanted in various regions of the cat brain and the long-term structural changes in the tissue surrounding the electrodes were studied by light and electron microscopy.

As observed by high magnification electron microscopy, potassium ions induce osmiophilia at the extracellular side of triple layered plasma membranes in the central nervous system of the rat. In contrast, sodium ions induce a widening of the intracellular lamina of these membranes, and do not result in observable extracellular deposits. The observations suggest the existence of potassium and sodium ion-selective structures associated with opposite surfaces of the neuron membrane. Use of the fixative vehicle as a cytochemical reagent is proposed.

Various alkali metal buffers were used in combination with glutaraldehyde, formaldehyde and acrolein to fix rat visual cerebral cortex by perfusion. Standard procedures of post-fixation, dehydration, and embedding were followed so that the buffer became the experimental variable. The cacodylate buffer system proved most versatile, and caesium, rubidium, potassium, sodium, and lithium cacodylate were used. An unusual amount of extracellular space averaging 300 nm was present with caesium cacodylate which contrasted with the apposed plasma membranes prevalent when lithium or sodium cacodylate was used. Rubidium and potassium cacodylate produced similar results with variable and irregular extracellular space. The effects of the addition of dimethyl sulphoxide, magnesium chloride and calcium chloride to the various cacodylate buffers were explored. In the case of caesium cacodylate, these additives caused less extracellular space and the retention of more material in this space in an irregular manner; with potassium cacodylate there was no noticeable difference, but with sodium and lithium cacodylate extracellular space increased. We suggest that the observed differences in extracellular space are explanable in terms of changes occurring in the polysaccharide cell coat which are caused by the buffers and additives.

A technique for computer reassembly and plotting of morphological serial sections in three dimensions is described. The computer reconstruction is obtained at a savings in cost and time compared to previous model-processing or isometric drawing methods.

Five bacteriophages were isolated from lysogenic strains of Salmonella potdam. On the basis of plaque morphology, thermostability, serology, host range, one-step growth parameters, and phage morphology, they were divided into three groups: group A, phages P4 and P9c; group B, phages P3 and P9a; and group C, phage P10. Group A phages had a hexagonal head 55 nm in diameter with a short tail 15 nm long. These phages were particularly characterized by high thermostability, lack of serological relationship with any of the other phages, and restriction of lysis to other Salmonella strains of Kauffmann-White group C1. Group B phages had a head identical in size and shape to that of the A phages, but they possessed a tail 118 nm long with a contractile sheath. A unique feature was the occurrence of tail fibers at the end of the core rather than at the base of the sheath. These phages were considerably less thermostable, had extended host ranges, and were serologically distinct from each other but unrelated to the A phages. The group C phage, P10, had a head identical to that of the A and B phages. It had a tail 95 nm in length, with tail fibers attached to a base plate at the end of a contractile sheath. P10 was highly sensitive to heat, lysed only smooth strains of Salmonella, and showed a degree of serological relationship to both B phages. The relationship of these phage groups to previous Salmonella phage grouping schemes is discussed.

SUMMARYThe production of vacuolated mitochondria and myelin figures can be markedly reduced with aldehyde perfusion fixation by perfusing a concentrated aldehyde solution before the conventional perfusate. A method is presented for accomplishing this with no practical time delay. Factors causing myelin figures and damaged mitochondria are discussed.Gum acacia helps prevent ‘washed‐out’ cells and processes, and seems to aid myelin preservation. A clear solution is necessary for a good perfusion. Techniques for accomplishing this are presented.A concentrated formaldehyde solution perfused as a small volume immediately prior to the conventional perfusate causes alterations of the endoplasmic reticulum, including the subsurface cisterns, and an increase in numbers of spine apparatuses. Evidence is presented by using tubes of gelatin containing Schiff's reagent that formaldehyde is the fastest penetrating aldehyde but that it only fixes about half the distance it penetrates. Acrolein penetrates more rapidly than glutaraldehyde but fixes only as far as formaldehyde. Glutaraldehyde fixes as far as it penetrates.

During the study of rat visual cortex by electron microscopy a neuron has been observed which shows a capillary passing through it. Cytoplasm of the neuron can be seen entirely encircling the capillary without any discontinuities. Sufficient serial sections of this peculiar association of structures were available to verify that the capillary is indeed penetrating the neuron, and that the relationship is not caused by grazing, tangential sections through a capillary bulging into a neuron. Moreover, a tangential section of a capillary would be quite obvious from the profile of the lumen and the appearance of the wall. That the cell surrounding the capillary is a neuron seems certain, for it exhibits typical neuronal structure, including synapses. Fine details of such a synapse are shown. At no level has the capillary been seen to be in direct contact with the neuronal plasma membrane, since there is present an intervening, and at times very thin and attenuated, layer of cytoplasm.

Hypertonic fixatives perfused well, but gross brain shrinkage was excessive with extreme hypertonicity. Increasing the amount of buffer in a perfusate to 1200 mOsM resulted in shrinkage of cells and large amounts of extracellular space. Increasing the fixative concentration did not cause extensive extracellular space formation but had only relatively subtle effects on fine structure. Moderate hypertonicity resulted in little gross brain volume change. Isotonic (300–320 mOsM) perfusates acted similarly to hypotonic fixatives, which caused gross swelling of the brain and poor preservation on the ultrastructural scale and would not flow well at perfusion. While extreme pH's (2 and 11) were detrimental to preservation of structure, pH's of 6 and 8 caused little change. A brief “wash” of pH 2.0 buffer immediately preceding pH 7.4 perfusion caused the appearance of large scale extracellular space, but also fair preservation of ultrastructure.

Journal of Comparative NeurologyVolume 122, Issue 2 p. 281-295 Article Macroglial identification in electron micrographs Robert L. Schultz, Robert L. Schultz Department of Anatomy, Loma Linda University, Loma Linda, California Supported by U.S. Public Health Grant no. B-1801.Search for more papers by this author Robert L. Schultz, Robert L. Schultz Department of Anatomy, Loma Linda University, Loma Linda, California Supported by U.S. Public Health Grant no. B-1801.Search for more papers by this author First published: April 1964 https://doi.org/10.1002/cne.901220210Citations: 34 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Literature Cited Achucarro, N. 1918 On the evolution of the neuroglia and especially their relation to the vascular apparatus. J. nerv. ment. Dis., 48: 333–342. Borysko, E. 1956 Recent developments in methacrylate embedding. I. A study of the polymerization damage phenomenon by phase contrast microscopy. J. biophys. and biochem. Cytol., Suppl., 4: 3–14. 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BY using glutaraldehyde and formaldehyde as primary in vivo fixatives for electron microscopy, we have found in this investigation a tightly apposed plasma-membrane relationship within grey matter of the adult mammalian central nervous system. The previously reported and widely accepted extracellular compartment of more than 100 Å in width1,2 has been questioned by Sjöstrand3. In the work reported here it was completely absent in most places and when present measured less than 100 Å in width.