Robert L. Schultz

Angiogenesis after stroke is correlated with increased numbers of macrophages: the clean-up hypothesis.

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.

Neuroprotection of Ischemic Brain by Vascular Endothelial Growth Factor Is Critically Dependent on Proper Dosage and May Be Compromised by Angiogenesis

Vascular endothelial growth factor (VEGF) is currently considered a potential pharmacologic agent for stroke therapy because of its strong neuroprotective and angiogenic capacities. Nonetheless, it is unclear how neuroprotection and angiogenesis by exogenous VEGF are related and whether they are concurrent events. In this study, the authors evaluated by stereology the effect of VEGF on neuronal and vascular volume densities of normal and ischemic brain cortices of adult male Sprague-Dawley rats. Ischemia was induced by a 4-hour occlusion of the middle cerebral artery. Low, intermediate, and high doses of VEGF165 were infused through the internal carotid artery for 7 days by an indwelling osmotic pump. The low and intermediate doses, which did not induce angiogenesis, significantly promoted neuroprotection of ischemic brains and did not damage neurons of normal brains. In contrast, the high dose that induced angiogenesis showed no neuroprotection of ischemic brains and damaged neurons of normal brains. These findings suggest that in vivo neuroprotection of ischemic brains by exogenous VEGF does not necessarily occur simultaneously with angiogenesis. Instead, neuroprotection may be greatly compromised by doses of VEGF capable of inducing angiogenesis. Stroke intervention efforts attempting to induce neuroprotection and angiogenesis concurrently through VEGF monotherapy should be approached with caution.

The impurities in commercial glutaraldehyde and their effect on the fixation of brain.

Evidence is presented that indicates the material absorbing at 235 nm in commercial glutaraldehyde is a dimer of glutaraldehyde having the form of an α, β-unsaturated aldehyde. If one desires to minimize polymerization, glutaraldehyde should be protected from exposure to volatile acids or bases and should be stored in dilute solution or at subfreezing temperatures. Barium carbonate should not be added to glutaraldehyde solutions. The presence of appreciable quantities of dimer in fixative solutions of glutaraldehyde for electron microscopy improves quality of structural preservation when compared with distilled glutaraldehyde fixation under identical conditions.

A modified aldehyde perfusion technique for preventing certain artifacts in electron microscopy of the central nervous system.

The 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.

The ultrastructure of the sheath around chronically implanted electrodes in brain

SummaryInsulated, 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.A sheath surrounded and separated the electrode from normal grey or white matter. A layer of foreign body giant cells of variable thickness was formed adjacent to the electrode. This layer was attenuated in some places so that it was unrecognizable by light microscopy. The bulk of the sheath structure consisted of collagen fibrils, leptomeningeal cells and hypertrophied astrocytes. Areas consisting of modified leptomeningeal cells with long thin processes we designated as spongy areas. These have not been previously reported using the electron microscope.Glycogen bodies were seen in leptomeningeal cells.Astrocytes became greatly enlarged and were more numerous in and around the sheath. Oligodendrocytes contained lamellar bodies, and direct continuity was shown between a lamellar body and an adjacent myelin sheath. Myelin was seen in abnormal sites (around oligodendrocytes and neurons) and in unusual configurations.Neuronal changes near the sheath included whorls and stacks of modified endoplasmic reticulum and the presence of cytoplasmic nucleolus-like bodies. Reactive, regenerative and degenerative axons were observed.Blood vessels were more numerous in the sheath and surrounding tissue than normal. Perivascular spaces were prominent even around capillaries and often plasma cells and monocytes were in these spaces. As compared to normal tissue the extracellular space is noticeably increased.Electrodes passing through ventricles were surrounded with a sheath covered with ependymal cells. This sheath was comparable in structure to the sheath present around the electrode in other locations.

Intranuclear inclusions in neurons of the cat primary olfactory system

Abstract The ultrastructure of two diverse types of intranuclear inclusions are described for neurons of the cat olfactory system. One is a rod or spindle-shaped structure of densely packed parallel filaments and the other has a crystalline lattice appearance. Apparently, both inclusions are more widespread in the olfactory brain than many other brain regions. Three-dimensional analysis was carried out using serial section methods to assess the solid geometry of the rodlet and lattice and the relationship of these intranuclear structures to the nucleolus and nuclear membrane. By this technique it is demonstrated that the rodlet crosses the nucleoplasm from pole to pole but does not penetrate the nuclear membrane. The lattice is also extensive in its intranuclear arrangement. From analytical measurements obtained from micrographs through different orientation planes of the lattice, an interpretative model is proposed that accords with all the various structural features of this extraordinary intranuclear body. The prevalence of both of these intranuclear inclusions in the cat anterior olfactory nucleus affords an opportunity for conducting cytochemical experiments attempting to understand the fundamental nature of these structures.

The oligodendroglial reaction to brain stab wounds: An immunohistochemical study

SummaryMyelin/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′-phosphodiesterasepositive 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.

Rapid changes of light microscopic indices of osteoclast-bone relationships correlated with electron microscopy.

SummaryThe 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 ultrastructure of the nerve fibers and pinealocytes in the rat pineal stalk.

In view of the increasing interest in the central innervation of the mammalian pineal gland, this aspect was studied in depth in the rat. This species is especially suited since the nerve fibers in question form a distinct bundle running from the deep to the superficial pineal gland through the pineal stalk. The axons were counted and analysed ultrastructurally in the pineal stalks cut transversely at three levels (proximal, intermediate, and distal) relative to the neural axis and in longitudinal sections. The number of nerve fibers was highly variable, ranging from 551 to 1, 132 proximally and from 110 to 448 distally, indicating that many fibers terminate in the stalk or leave the stalk after forming a loop. Large myelinated axons, which are abundant proximally, appear to lose their sheaths along their course through the stalk. Most of the axons were small and unmyelinated. A few of these had the appearance of sympathetic fibers and disappeared after sympathectomy. Others contained abundant neurosecretory granules, and, according to the literature, may originate in the hypothalamic paraventricular nuclei. The majority of the small axons which are apparently devoid of granules and dense‐cored vesicles may come from the habenular nuclei and the stria medullaris. In addition to axons, the stalk contains astrocytes, a few oligodendrocytes and Schwann cells, as well as pinealocytes identical to those of the superficial pineal gland.

Computer Graphics in Three Dimensions for Perspective Reconstruction of Brain Ultrastructure

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.