Marshall L. Rennels

Scanning Electron Microscopic Observations on the Luminal Surface of the Rabbit Common Carotid Artery Sublected to lschemia by Arterial Occlusion

The scanning electron microscope (SEM) has been employed to study the effects of ischemia on the luminal surface of the common carotid artery. Fifteen adult rabbits were lightly anesthetized and the common carotid arteries surgically exposed. The right carotid artery was occluded with a single Heifetz clip for five minutes (five animals), 15 minutes (five animals), and 30 minutes (five animals). Following removal of the clip, the animals were immediately perfused with glutaraldehyde and the arteries excised and prepared for critical point drying. Four additional rabbits were perfused following the same method with no surgical procedures performed in the neck. Normal aortas were also examined. The nature and frequency of endothelial cell alterations were determined by analysis of ten randomly selected SEM fields. Examination of the endothelial surface of arterial segments distal to the occluding clip revealed the presence of numerous “crater-like” defects as well as outpouchings or “balloons.” The numbers of craters and balloons were significantly increased in the ischemic (distal) arterial segment as compared to either proximal or sham-operated control segments (P < 0.001). These endothelial cell alterations were never observed in random micrographs of arterial segments taken from unoperated control animals, but were seen at the ostia of some intercostal arteries of the aorta. It is suggested that these craters and balloons could cause interference with blood flow and the for mation of platelet thrombi by their protrusion into the lumen, as well as alteration of the permeability of the arterial intima.

Caveolar systems and sarcoplasmic reticulum in coronary smooth muscle cells of the mouse.

Vascular smooth muscle cells in mouse heart contain prominent membrane systems (“sarcotubules”). One of these systems consists of vesicular structures whose unit membranes are continuous with the sarcolemma and which occur either as single caveolae, more complex tubules, or branched chains of fused caveolae. Such caveolar systems are both analogous to, and homologous with, the T or T-axial tubular systems of striated muscle cells. A second system of membranes, the sarcoplasmic reticulum (SR), comprises tubules and saccules that often come into close association with the sarcolemma but apparently are not open to the extracellular space. In addition to forming “couplings” with the sarcolemma, the SR often comes into close contact with mitochondria and caveolae. The ultrastructural complexity of these membrane systems in coronary vascular smooth muscle equals or surpasses that of smooth muscle cells in the large blood vessels that have been extensively studied by other investigators.

Preparation of vascular endothelium for scanning electron microscopy: a comparison of the effects of perfusion and immersion fixation

A comparison of currently used methods of tissue fixation for scanning electron microscopic study of vascular endothelium revealed that in situ fixation by intravascular perfusion is superior to immersion for the preservation of endothelial surface morphology.

Endothelial Cell Ischemic Injury: Protective Effect of Heparin or Aspirin Assessed by Scanning Electron Microscopy

Scanning electron microscopic observations of the luminal surface of the rabbit common carotid artery subjected to occlusion for 30 minutes or two hours revealed crater-like and balloon-like defects in the endothelial surface. The frequency of occurrence of these abnormalities was significantly decreased by pretreatment with heparin or aspirin in doses considered to have antiplatelet aggregating activity.

A method for microscopic studies of cerebral angioarchitecture and vascular- parenchymal relationships, based on the demonstration of ·paravascular’ fluid pathways in the mammalian central nervous system

A new method is described for morphological studies of blood vessels and related cellular elements in the mammalian central nervous system (CNS). The tracer protein, horseradish peroxidase (HRP), in solution, is infused intraventricularly or intracisternally in anesthetized animals over 5-10 min. During this period, HRP in the subarachnoid space enters the perivascular spaces around penetrating arterioles and rapidly permeates the gliovascular basal laminae surrounding capillaries. After fixation by intravascular perfusion of aldehydes, brain sections are incubated with the highly sensitive chromogen, tetramethylbenzidine. Intraparenchymal blood vessels throughout the CNS are vividly demonstrated for light microscopy by HRP reaction product in their perivascular spaces or basal laminae. Correlative ultrastructural investigations of specific blood vessels and related parenchymal elements can be conducted using adjacent sections.

Innervation of Capillaries by Local Neurons in the Cat Hypothalamus: A Light Microscopic Study with Horseradish Peroxidase

The protein tracer horseradish peroxidase (HRP) has been used in an attempt to define the cell bodies of origin of “nonadrenergic” varicose axons which terminate on the walls of hypothalamic capillaries. Capillaries in this region are also known to receive direct axonal contacts from adrenergic neurons in the pontine locus ceruleus. Solutions of HRP were infused into the lateral ventricles of adult cats of either sex and permitted to circulate in the cerebrospinal fluid spaces for 10 min, 20 min, or 2 h. During these periods HRP entered the perivascular spaces around penetrating arterioles and spread into the surrounding extracellular spaces of the hypothalamus. Certain neurons in the periarteriolar neuropil were consistently labeled by the tracer after all three circulation periods. These cells, including all of their processes, could be visualized in detail. Most neurons, by contrast, did not accumulate HRP. The axons of some tracer-filled neurons terminated on the walls of capillaries in the immediate vicinity of the penetrating arteriole. The arrangement and distribution of these cells suggest that they may provide a substrate for local neural influences on the hypothalamic microcirculation.

Delivery of Solutes in Cerebrospinal Fluid to Central Neurons via “Paravascular” Fluid Pathways in the Central Nervous Systema

Blood vessels throughout the brain and spinal cord are surrounded by longitudinal fluid pathways that are in continuity with cerebrospinal fluid (CSF) in the subarachnoid space (SAS).’.’ These pathways can be demonstrated light microscopically by infusion of the tracer protein, horseradish peroxidase (HRP), into the SAS and subsequent localization of the enzyme in brain sections using the highly sensitive chromogen, tetramethylbenzidine (TMB)? HRP enters the perivascular spaces (PVS) around penetrating arterioles and, within minutes, is distributed throughout the CNS along the basal laminae (BL) of capillaries. This rapid, unidirectional fluid/tracer movement along the vascular network appears to be facilitated by arteriolar pulsations? From these “paravascular pathways,” HRP spreads into the tissue spaces and also selectively delineates localized groups of neurons. A needle was inserted into the cisternae magna of adult cats anesthetized with sodium pentobarbital, and CSF elllux was measured. An equal volume (0.5-1.0 ml) of 4.0% HRP (Sigma Type 11) in Hanks’ balanced salt solution was infused by gravity flow. After HRP circulation periods of 4 rnin (6 cats) or 10 min (9 cats), fixation was camed out by perfusion of aldehyde solutions? Vibratome sections (100 pm) of the forebrain and brainstem were prepared and HRP was localized using TMB3 or diaminobenzidine as substrates. CNS intraparenchymal vessels were outlined, in toto, by paravascular reaction product after 10 min HRP circulation in the SAS, with TMB incubation. In the brainstem, vascular outlining occurred after as little as 4 min HRP circulation, and localized groups of tracer-containing neurons were observed in the hypothalamus and in lateral tegmental zones of the midbrain, pons and medulla (FIG. 1). This rapid cellular uptake was not observed elsewhere. The soma or dendrites of HRP-filled cells