John Beggs

Ultrastructure of three choroid plexus papillomas

This report describes the ultrastructure of three choroid plexus papillomas (CPP) from female patients, aged 2 months, 3 years, and 57 years, respectively. The infantile case manifested clinical evidence of excess CSF formation. All three were well‐differentiated neoplasms resembling normal choroid plexus (CP). Each displayed distinctive individual patterns of plasmalemmas, organelle population, and nuclei. The two childhood tumors contained prominent deposits of cytoplasmic glycogen, thus simulating the embryonic state. The vasculature of all three lesions included fenestrated capillaries indistinguishable from those of normal CP. Ventricular plasmalemmas were separated from lateral and basal plasmalemmas by tight junctions. The geometrical relationship between capillaries, tumor cells, and ventricle was characteristic of epithelia involved in fluid transport. These findings offer structural evidence that well‐differentiated CPPs are capable of participating in CSF formation.

Transperineurial Arterioles in Human Sural Nerve

The perineurial sheath of nerve fascicles is a protective cellular layer that separates the endoneurium from the epineurium. Transperineurial arterioles (TPA) connect the endoneurial capillary plexuses to the epineurial arterial nutrient supply. Transperineurial arterioles are defined as any arteriole that is confined to a perineurial cell compartment, which would include all arterioles within the perineurium proper or within perineurial sleeves in the epi- or endoneurium. In this study of biopsied human sural nerves, three-dimensional reconstruction of one μm sections and ultrastructural analysis of step serials, we find that TPA are confined in open-ended perineurial sleeves by which they pass from the epineurium through the perineurial sheath proper into the endoneurium. Most TPA are terminal arterioles as evidenced by size (10–25 μm), morphological characteristics, and the fact that they connect with capillaries. Transperineurial arterioles gradually lose their continuous muscle coat and become post-arteriolar capillaries (PAC). Vascular segments that emerge into the endoneurium from the perineurial sleeves are generally of the PAC variety. Transperineurial arterioles and post-arteriolar capillaries are often associated with a plexus of unmyelinated nerve fibers. Axon varicosities exhibit a variety of morphologically distinct vesicles including dense-cored and a diversity of agranular vesicles. These findings suggest that TPA play a role in the neurogenic control of endoneurial blood flow.

Innervation of the Vasa Nervorum: Changes in Human Diabetics

Transperineurial and epineurial vessels are innervated by plexuses of unmyelinated axons. Human sural nerve biopsies were examined ultrastructurally and immunocytochemically with an antibody which recognizes a neuronal and neuroendocrine protein, PGP 9.5, to characterize perivascular axons of these plexuses. Diabetics exhibited a greater degree of abnormal innervation of the vasa nervorum than nondiabetics with and without neuropathy. Abnormal innervation included: a reduction in the percentage of vessels exhibiting perivascular axons and a concomitant increase in the percentage of vessels having denervated Schwann ceil units, particularly around vessels confined to perineurial compartments, and remaining axons in nerves from diabetics exhibited fewer varicosities. Denervated arterioles of diabetics also displayed structural changes indicating injury. The aneriolar structural, defects and loss of neurogenic control of neural blood flow may lead to or aggravate endoneurial ischemia or hypoxia. The patchy, focal endoneurial fiber loss that is prominent in proximal nerves and associated with the distal myelinated fiber loss of some diabetic patients may be due in part to perivascular denervation of the vasa nervorum.

Comparative Study of Neuroma Formation in the Rat Sciatic Nerve after CO2 Laser and Scalpel Neurectomy

Recent reports have suggested that peripheral neurectomy with the CO2 laser may be effective in preventing subsequent neuroma formation. To study this question further, we performed bilateral sciatic nerve sections in 31 rats using a steel scalpel on one nerve and a CO2 laser on the opposite side. The animals were killed 30 days after neurectomy and specimens were removed for gross observation, light microscopy, and electron microscopy. Axon composition studies were carried out in selected animals and axon diameter histograms were prepared. Well-formed neuromas were present in nerves transected by both the scalpel and the CO2 laser. The scalpel neuromas were characterized by a greater degree of interfascicular collagen deposition and perineurial cell proliferation. Laser neuromas demonstrated less perineurial compartmentalization and scar tissue formation. In addition, a foreign body reaction with multinucleated giant cells surrounding carbonaceous debris was seen that was not present in the scalpel neuromas. Analysis of axon composition studies revealed that both neuromas had a greater density of axons and a higher percentage of small diameter myelinated and unmyelinated axons as compared to control nerves. Laser neuromas had more axons per unit area than scalpel neuromas, but the percentage composition of axons was very similar in the two groups. We could find no evidence in the rat sciatic nerve model that CO2 laser neurectomy is less likely to result in neuroma formation than is conventional scalpel neurectomy.

Unmyelinated Nerve Fiber Estimation by Immunocytochemistry. Correlation with Electron Microscopy

Electron microscopy (EM) is currently required for quantitation of unmyelinated nerve fiber (UMNF) densities. Electron microscopy is time-consuming, costly, and generally only considers a fraction of an entire nerve. Anti-PGP 9.5, which recognizes a neuron-associated antigen, may be used in glutaraldehyde:fixed, paraffin-embedded human sural nerve biopsies to identify unmyelinated axons. In nerves counterstained with Luxol fast blue, the correlation between EM-obtained UMNF densities and paraffin-obtained UMNF densities was excellent (p<0.0001). In addition, myelinated nerve fiber (MNF) densities estimated by the same method from paraffin-embedded nerve gave excellent correlation with traditional morphometric estimates (p=0.0026). PGP 9.5 immunocytochemistry enables the detection of minute axons (<0.5µm) and multiple axons per Schwann cell subunit, but does not allow for the preparation of accurate fiber size histograms or the analysis of UMNF pathology. Whether used for UMNF quantitation or as a qualitative review of the UMNF population of a nerve, this method is quicker and less expensive than traditional EM methodologies.

Relationships between microvascular function and capillary structure in diabetic and nondiabetic human skin

Despite the commonly held view that abnormalities in capillary morphology, in particular thickening of the capillary basement membrane, are partly responsible for diabetic ischemia, few studies have correlated anatomic and hemodynamic variables in the same diabetic subjects. In a previous study of 24 type II (non-insulin-dependent) diabetic subjects and 24 agematched control subjects, we showed that a standard finger exercise vasodilated cutaneous forearm vessels nearly equally (51%), but the postarteriolar flow responded differently between groups. Nondiabetic subjects increased flow by recruitment of capillaries, whereas diabetic subjects did so by capillary flow augmentation. Moreover, resting permeability-surface area product (PS) to pentetic acid was 85% higher in diabetic than nondiabetic subjects. In this study, these same subjects had their forearm skin biopsied and examined morphometrically by electron microscopy for capillary radius, basement membrane thickness, endothelial cell density, and a folding index of luminal membrane reduplication. All morphological variables were correlated stepwise in a saturated, analysis of covariance model with the physiological results. The correlations were sparse and specifically excluded basement membrane thickness. The highest r2 value was .432 between resting PS and a ratio of capillary density to endothelial cell number per capillary. These studies show little evidence that diabetic microvascular physiological variables are tightly connected to morphometric changes except for minor permeability changes, which rise with capillary density and decrease with endothelial cell number. Because PS to pentetic acid is increased in diabetic subjects at any level of capillary density, it seems reasonable that permeability may be increased above that of nondiabetic subjects. However, such a conclusion is tentative because anatomic measurement of capillary density gives only the maximum estimate of capillaries in the tissue, which may be greater than the number of capillaries with flow at the time of physiological measurement.

Hypothalamic hamartomas associated with epilepsy: ultrastructural features.

Hypothalamic hamartomas (HHs) are associated with a catastrophic form of childhood epilepsy and are intrinsically epileptogenic for the gelastic seizures that are peculiar to this disorder. The cellular mechanisms of seizure generation within HH tissue are unknown. Hypothalamic hamartoma tissue consists of well-differentiated neurons interspersed with glial cells. Based on intrinsic electrophysiologic properties, we hypothesized that small and large HH neurons would have different ultrastructural features. Surgically resected HH tissue samples from 7 patients with refractory epilepsy were studied using electron microscopy. Models of neurons were made from reconstruction of serial sections. Nissl body density, polyribosome density, glycogen density, and the ratio of nuclear area to cytoplasmic area differed significantly between small and large neurons. Abundant small neurons were found in clusters and elaborated terminals that made symmetric, putatively inhibitory, synapses. Symmetric synapses were found predominantly on the soma and proximal dendrites of large projection-like neurons, whereas asymmetric, putatively excitatory, synapses were found primarily on distal dendrites. All samples showed unusual dendritic varicosities. The presence of these morphologically and functionally distinct neurons, the large number of nerve fibers in the neuropil, and the presence of inhibitory and excitatory synapses suggest that HH tissue has the necessary substrate to generate seizures.

Comparison of Measurement Methods for Capillary Basement Membrane Thickness: Collapsed-Ellipse Technique

Both the grid method of Siperstein et al. (tSIP) and the minimum-points method of Williamson et al. (tWIL) for measurement of capillary basement membrane thickness are inaccurate for assessing mean true membrane thickness of a given section (tTRUE) for various reasons, including errors in selectivity, sensitivity, and geometry. In general, it is agreed that tSIP > tTRUE > tWIL, but estimates of tTRUE beyond this have not been made. In this study, a collapsed-ellipse method for approximating tTRUE is presented that measures thickness by areas (tEA). One hundred-twenty capillaries from the forearm skin of 12 diabetic subjects and 12 age-matched controls were measured to examine these concepts. We found that, whereas tWIL was up to 63% below tTRUE, tEA was 30% too low. Although tSIP, tWIL, and tEA did not distinguish between diabetic and normal subjects, tEA and tWIL measurements had highly predictable and small errors, and tSIP had unpredictable ranges of error, especially when tSIP was low.

Gap Junctions Contribute to Ictal/Interictal Genesis in Human Hypothalamic Hamartomas

Human hypothalamic hamartoma (HH) is a rare subcortical lesion associated with treatment-resistant epilepsy. Cellular mechanisms responsible for epileptogenesis are unknown. We hypothesized that neuronal gap junctions contribute to epileptogenesis through synchronous activity within the neuron networks in HH tissue. We studied surgically resected HH tissue with Western-blot analysis, immunohistochemistry, electron microscopy, biocytin microinjection of recorded HH neurons, and microelectrode patch clamp recordings with and without pharmacological blockade of gap junctions. Normal human hypothalamus tissue was used as a control. Western blots showed increased expression of both connexin-36 (Cx36) and connexin-43 (Cx43) in HH tissue compared with normal human mammillary body tissue. Immunohistochemistry demonstrated that Cx36 and Cx43 are expressed in HH tissue, but Cx36 was mainly expressed within neuron clusters while Cx43 was mainly expressed outside of neuron clusters. Gap-junction profiles were observed between small HH neurons with electron microscopy. Biocytin injection into single recorded small HH neurons showed labeling of adjacent neurons, which was not observed in the presence of a neuronal gap-junction blocker, mefloquine. Microelectrode field recordings from freshly resected HH slices demonstrated spontaneous ictal/interictal-like discharges in most slices. Bath-application of gap-junction blockers significantly reduced ictal/interictal-like discharges in a concentration-dependent manner, while not affecting the action-potential firing of small gamma-aminobutyric acid (GABA) neurons observed with whole-cell patch-clamp recordings from the same patients HH tissue. These results suggest that neuronal gap junctions between small GABAergic HH neurons participate in the genesis of epileptic-like discharges. Blockade of gap junctions may be a new therapeutic strategy for controlling seizure activity in HH patients.

The Pathophysiology of Bacterial Meningitis

Bacterial meningitis is the most common type of central nervous system (CNS) infection. Most recent reviews of the subject quote an incidence of five per 100,000 with the majority of cases appearing in the pediatric age group.1–4 Approximately 25,000 cases occur annually in the U.S. The mortality rate is 15 percent and the morbidity rate is at least twice this figure.5