Assessing quantitative chimerism longitudinally: technical considerations, clinical applications and routine feasibility.

In this review, we describe the current laboratory approach to quantitative chimerism testing based on short tandem repeats (STRs), focusing on a longitudinal analysis. The latter is based on relative changes appearing in the course of sequential samples, and as such exploits the ultimate potential of this intrinsically semiquantitative platform. Such an analysis is more informative than single static values, less likely to be confused with platform artifacts, and is individualized to the particular patient. It is particularly useful with non-myeloablative conditioning, where mixed chimerism is common. Importantly, longitudinal monitoring is a routinely feasible laboratory option because multiplex STR-polymerase chain reaction kits are available commercially, and modern software can be used to perform computation, reliability testing and longitudinal tracking in a rapid, easy to use format. The ChimerTrack application, a shareware, user friendly program developed for this purpose, produces a report that automatically summarizes and illustrates the quantitative temporal course of the patient's chimeric status. Such a longitudinal perspective enhances the value of quantitative chimerism monitoring for decisions regarding immunomodulatory post transplant therapy. This information also provides unique insights into the biological dynamics of engraftment underlying the fluctuations in the temporal course of a patient's chimeric status.

Morphological responses to local CNS trauma: sprouting and synaptogenesis within membranes implanted into mature cerebral cortex of the rat.

The purpose of this study was to document in vivo neuronal sprouting in response to local trauma of the mature cerebral neocortex. The experimental approach enabled direct and unequivocal ultrastructural identification of newly sprouted neuronal processes and their synaptic relationships. Morphologically detectable factors that influence neuronal sprouting were also assessed. Tiny fragments of porous, synthetic membrane were implanted into the mature neocortex of the rat. After one week, axons, dendrites, growth cone-like structures, synapses, and processes from astrocytes and microglia were present deep within the interstices of the implant. Morphometric analysis of synapses within the implant, support the qualitative impression of active neuronal ingrowth and de novo synaptogenesis by these sprouted elements. Astrocytic processes appear to comprise the major barrier to neuronal invasion of the implant. Several observations suggested that glia sometimes may facilitate neuronal ingrowth. These findings provide direct morphological documentation of axonal and dendritic plasticity in the mature rat neocortex. The work demonstrates a strategy that could be useful for further evaluating the cellular mechanism of in vivo neuronal regeneration.

Development of neocortical circuitry: histochemical localization of acetylcholinesterase in relation to the cell layers of rat somatosensory cortex.

Acetylcholinesterase (AchE) was histochemically localized in neocortex cerebri of newborn to 1-week-old rats. At birth AchE-dependent staining is limited to scattered somata (Cajal-Retzius cells) in the marginal zone and a few fibers and somata in primordial layer VI. By the end of the first week, neuronal elements are relatively well stained in particular cell laminae, giving the appearance of three horizontal AchE-rich "bands" which alternate with AchE-poor laminae. The subpial band (layer I) is a narrow tangential zone of intensely staining fibers and scattered somata. The mid-cortical band contains an AchE-positive fiber plexus (primordial layer IV) and numerous stained somata (pyramids of primordial layer V). In layer IV of the SmI region, intermittent foci of staining are noted which overlap the barrels' distribution in the barrel field. The deep cortical band (bottom of primordial layer VI) consists of numerous stained somata (Martinotti cells). It is concluded that there is a laminar pattern of acetylcholinesterase-dependent staining in postnatal rat somatosensory cortex, and that the laminar pattern bears a consistent spatial relation both to the cell layers and to the depths of high synapse density in immature cortex.

Myelopathic neurosarcoidosis: diagnostic value of enhanced MRI.

Neurosarcoidosis is an underdiagnosed variant of the systemic disease. We report a case of myelopathic neurosarcoidosis, noting the contribution made by MRI in establishing the diagnosis, and we discuss the possibility that the disease is differentially responsive to various steroid formulations.

Acetylcholinesterase in immature thalamic neurons: relation to afferentation, development, regulation and cellular distribution.

The transient appearance of intense acetylcholinesterase reactivity in some immature, noncholinergic neurons has not been adequately explained. In this study two questions were investigated that relate to several possible roles for acetylcholinesterase. First, what factors influence the onset and maintenance of reactivity? Second, what are the temporal and spatial features of the cellular expression in relation to stages of neuronal development? Using light- and electron-microscopic histochemical methods, the non-cholinergic ventrobasal complex in thalamus of the immature rat was examined. Ultrastructural observations on fetal ventrobasal complex demonstrated that the onset of acetylcholinesterase reactivity precedes ingrowth of most extrinsic afferents. These inputs are, therefore, unlikely to provide the signal for onset. In transplants and explants, acetylcholinesterase persisted in ventrobasal complex neurons independent of their principal afferents. However, afferentation can affect reactivity. The patterned variation in intensity, characteristic of infant ventrobasal complex, was dramatically altered by unilateral interruption of its afferentation. The changes in intensity patterning could reflect changes in acetylcholinesterase metabolism, since postnatal treatment with an irreversible inhibitor (diisofluorophosphate) in vivo demonstrated resynthesis of acetylcholinesterase. The period of peak intensity of acetylcholinesterase reactivity normally began abruptly at 18 days of gestation +/- 12 h and continued until 4-6 days postnatally. This period follows neurogenesis and migration, but precedes active synaptogenesis. It coincides with outgrowth and initial contacting of cell processes in the ventrobasal complex. The timing complements the ultrastructural finding that acetylcholinesterase-dependent reaction product most commonly is localized to small patches of surface membrane, where distal processes contact each other, non-synaptically. Together these data suggest three points. First, that the expression of acetylcholinesterase in the immature ventrobasal complex neuron is probably under active metabolic control, responsive to both intrinsic and environmental factors. Second, that acetylcholinesterase expression is unlikely to result from a transient cholinergic input. Third, that the temporal and spatial characteristics of histochemical reactivity enable exclusion of several previously suggested explanations for the occurrence of acetylcholinesterase in the ventrobasal complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholinesterase in the ventrobasal thalamus: transience and patterning during ontogenesis.

In this study, maturational alterations in acetylcholinesterase-dependent staining of the thalamic ventrobasal complex of rat and mouse were examined. The study was undertaken to address the question of whether this nucleus exhibits transient acetylcholinesterase positivity during its development and whether the enzyme is likely to be synthesized by its immature intrinsic neurons. Also, the patterning due to acetylcholinesterase staining of cells and fibers, and the developmental changes in these patterns, have not been described in earlier work. In contrast to surrounding thalamic nuclei, the ventrobasal complex is acetylcholinesterase-positive at birth. In rat, acetylcholinesterase staining of the ventrobasal thalamus is still more intense than in adjacent nuclei at the end of the first week postnatally. Virtually all somata in the nucleus are filled with dense reaction product at this time. Ultrastructurally, reaction product is associated with the granular endoplasmic reticulum. At this stage, there is a marked difference in intensity of staining between the medial and lateral subdivisions of the nucleus, and patterned clustering of somata within each subdivision is readily appreciated in acetylcholinesterase-stained material. In the second postnatal week, intrinsic acetylcholinesterase activity is progressively lost. By the end of the third postnatal week, the nucleus is quite pale except for one area. In the posterior portion of the lateral subdivision, adjacent to the nucleus reticularis, interconnecting bundles of acetylcholinesterase-positive fibers enter the nucleus. They course medially in the lateral subdivision and break up into a plexus of fine fibers. The development of acetylcholinesterase-dependent staining patterns in the mouse is quite similar, except that histochemically detectable levels of enzyme are substantially lower in the neonatal period. It is concluded that the ventrobasal complex can be distinguished from other thalamic nuclei in regard to earlier onset and/or transience of acetylcholinesterase staining. Ultrastructural observations suggest that virtually all immature ventrobasal neurons are synthesizing acetylcholinesterase. It is suggested that the transient staining for enzyme is due primarily to alteration in synthesis and/or turnover in neurons of the ventrobasal complex. In addition, the acetylcholinesterase staining reveals a patterning of fibers and cells that also undergoes developmental alteration. Evidence is discussed suggesting that axons in the barrels of somatosensory cortex (SmI) are derived from these transiently acetylcholinesterase-positive somata. Consequently, the loss of acetylcholinesterase fiber staining in the barrels, during the third postnatal week (noted previously), may be related to a decrease in synthesis of enzyme in the neuronal somata of the ventrobasal complex.

Colonic aberrant crypts may originate from impaired fissioning: relevance to increased risk of neoplasia.

Colonic aberrant crypt foci (ACF) can be identified on the unembedded mucosal surface as clusters of abnormal crypts with enlarged, surface opening. Because dysplasia is frequent, and may be a precursor of carcinoma, epithelial changes have been well studied. However, the basis for the distinctive changes in crypt architecture remain unclear. We hypothesized that some of the architectural alterations of aberrant crypts may reflect impaired fissioning during normal crypt duplication cycles. Fissioning begins at the crypt base. Using morphometric and immunocytochemical approaches, we examined 55 human ACF, both dysplastic and nondysplastic, for their architectural features. Non-ACF mucosa was compared. Microscopically, all lesions contained crypts that were attached, paired, dilated, and angulated. In 3 dimensions, these features related to multiple, individual complexes of connected crypts, referred to as connected crypt structures (CCSs). CCSs terminated in enlarged surface openings (2 to 5 x normal) which are morphometrically equivalent to the macroscopic aberrant crypts (P > .1). These openings trap marker dye. Support for an origin of CCSs in impaired basal fissioning is 3-fold. Crypt profiles in ACF are twice as frequent in basal mucosa as superficially (P < .001); in normal mucosa, the ratio is 1. In a CCS with vertically connected, co-planar crypts, the upper parent crypt diameter was the sum of diameters of inferiorly attached daughter crypts (P > .1). Proliferating cell marker, Ki-67, is not expressed at attachment points. In non-ACF mucosa, isolated CCSs consistently occur at foci of mechanical crypt distortion such as mucosal folds. We conclude that a CCS is a fundamental component of ACF of all histotypes. Impairment of normal crypt fissioning is probably a major factor in the histogenesis of CCSs, which often occurs in settings of mechanical distortion of the mucosa. The pathological significance of this process may be in the formation of enlarged crypt openings. The latter could trap dietary carcinogens as they trap dye, and thereby predispose the CCS to dysplasia.

Waldenström's macroglobulinemia associated with AA amyloidosis.

It is widely accepted that amyloidosis in Waldenström's macroglobulinemia (WM) is exclusively due to amyloid light-chain deposition. However, only a small number of previous reports have actually characterized the type of amyloid in WM. We now report the third patient with WM and amyloid A protein (AA) amyloidosis. This patient developed malabsorption, nephrotic syndrome, and orthostatic hypotension. AA was immunohistochemically demonstrated in the rectal biopsy. In conjunction with previous examples of AA amyloidosis, the present report raises the possibility that AA amyloidosis may also occur in WM patients.

Neuronal differentiation in somatosensory cortex of the rat. I. Relationship to synaptogenesis in the first postnatal week.

Newborn (P-0 and P-1) through 6-day-old (P-6) rats were studied using light microscopic (Golgi) and ultrastructural methods. Previous studies demonstrated that early-formed synapses are concentrated at specific cortical depths, i.e. in strata. The present study shows that the synaptic stratum in the marginal zone corresponds to a dense fiber plexus and few somata (Cajal-Retzius cells). Axons in this zone almost exclusively form synapses on distal branches of dendrites originating in deeper lamina. In newborn neocortex there is a second synaptic stratum located deep to the cortical plate. It contains numerous axosomatic and axoproximal dendritic synapses as well as the most highly differentiated somata and proximal dendrites. By age P-6 there are 3 synaptic strata; one each in the marginal zone, cortical plate and 'subplate' layers. For all 3 strata a neuron's most differentiated dendrites are directed towards, traverse or run within, the nearest synaptic stratum. We conclude that, throughout the first postnatal week, the most mature dendrites of a given neuron generally occur at depths where synapse density is highest. At P-0 the most mature somata are similarly related to synaptic density.

Development of neocortical circuitry: quantitative ultrastructural analysis of putative monoaminergic synapses.

The distribution, numbers and morphology of presumed monoaminergic (MA) synapses were examined in somatosensory cortex of neonatal rats and mice (newborn to 16 days of age). MA synapses were identified using an ultrastructural cytochemical marker, 5-hydroxydopamine (5-OHDA), which results in the appearance of small granular vesicles (SGV) in their presynaptic terminals. From birth to 7 days of age, 20--30% of all synapses sampled in somatosensory cortex contain SGVs. However, few SGV synapses are seen in 8-day-old cortex and by 12 days of age, SGVs are no longer detectable in cortex. A specific laminar distribution for these SGV synapses -- which is distinct from the overall synaptic distribution -- is first seen at 3 days of age and is essentially unchanged until 7 days postnatally. During this entire period, the SGV synapses predominate in the primordium of layer IV, where they account for 50--70% of all synapses. Morphometric analysis of SGV synapses indicates that there are differences in junctional symmetry, vesicle shape and configuration of the contact zone between SGV and non-SGV synapses, as well as between SGV synapses themselves in the various cortical layers. The laminar distribution and morphological characteristics of SGV synapses suggest that the MA projection to neocortex exhibits a high degree of spatial specificity during its ingrowth. Also, the relatively high proportion of SGV synapses in the first postnatal week may reflect a potent influence exerted by the MA inputs on immature neocortex. The decreased numerical density of SGV synapses after 7 days of age is probably due to the development of the blood-brain barrier to 5-OHDA.

High density of cholinergic muscarinic receptors accompanies high intensity acetylcholinesterase-staining in layer IV of infant rat somatosensory cortex.

In this study the laminar localization of binding sites for the muscarinic radioligand [3H]3-quinuclidinylbenzilate was studied in somatosensory cortex of infant rat. Using light autoradiographic techniques, [3H]QNB was found to be concentrated in layer IV. Atropine-pretreated pups showed evenly dispersed, nonspecific binding in all layers. We conclude that cholinergic muscarinic receptors are concentrated in layer IV of infant rat somatosensory cortex. It is also in this lamina that an acetylcholinesterase-rich pathway terminates.

Basal forebrain innervation of rodent neocortex: studies using acetylcholinesterase histochemistry, Golgi and lesion strategies.

Acetylcholinesterase (AChE)-rich projections from basal forebrain to neocortex cerebri were characterized in the present study. The purpose was to investigate 3 aspects of these projections in rats and mice that have been incompletely described in previous work: intracortical organization of the fibers, subcortical pathways and axonal branching patterns of individual basal forebrain neurons. AChE histochemistry, lesions and Golgi impregnations were the principal strategies employed in this light microscopic study. The moderately dense, AChE-stained innervation of neocortex can be altered by intracortical lesions. The results depended on the region involved and the orientation of the lesion. Sagittal knife cuts had barely detectable effects, regardless of sites. Coronal knife cut lesions in medial cortex resulted in substantial loss of staining in cingulate and medial occipital fields. In contrast, coronal lesions of lateral or anterior cortex produce only small zonal reductions in staining. The interpretation of the latter findings that we favor is that AChE-rich basal forebrain fibers enter lateral/anterior cortex and branch densely there, but in tangentially limited and overlapping terminal domains. Observations on the topography and targets of AChE-rich basal forebrain cortical afferents revealed that the fibers could be grouped based on certain characteristics. Three sets of fibers were distinguishable: anterior pathway innervating cortex of the frontal pole. These fibers were traceable to the region of the substantia innominata/nucleus basalis. They crossed the neostriatum and external capsule in the sagittal plane, forming in 3 dimensions an orderly sheet-like array of fibers bridging the anteroventral surface of the neostriatum with nearby polar cortex medial pathway innervating cingulate and medial occipital cortex. Emerging predominantly from the region of the diagonal band, the fibers run caudally as a triangular bundle in deep layer VI of cingulate cortex. lateral pathway innervating most of remaining lateral neocortex. The fibers radiate out from substantia innominata/nucleus basalis with a complex 3-dimensional organization. In all pathways, fibers enter and initially run within layer VI before ascending pialward, although the intracortical course in layer VI differs between pathways. These fibers primarily terminate in layer V with a secondary concentration in layer I. However, the latter appears to receive substantial AChE-stained inputs from other sources, possibly intracortical, as well. The pathways overlap at their respective boundary zones. This system is comparably organized in rats and mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Somatosensory cortex: acetylcholinesterase staining of barrel neuropil in the rat.

Acetylcholinesterase (AchE) staining of layer IV of rat somatosensory (SmI) cortex was studied. In the barrel field of SmI, there are periodic, intensely AchE staining foci in the pattern and with the dimensions of barrels. The onset of this staining, at age 3 days, corresponds with the arrival of thalamocortical input to the barrels. Undercutting of SmI prevents staining in layer IV. We conclude that there are AchE-rich zones in layer IV that coincide with the specific thalamocortical projection to SmI.

The fine structural demonstration of monoaminergic synapses in immature rat neocortex.

The radial distribution of monoaminergic (MA) synapses has been studied in lateral neocortex (SI) of infant rats using an electron microscopic histochemical technique. Systemically administered 5-hydroxydopamine crosses the blood-brain barrier and produces small granular vesicles (SGV) in 30% of all synaptic terminals in SI. In the primordium of layer IV, at six days postnatal, 70% of the synaptic terminals contain SGVs. Immature cortex thus contains a high proportion of synapses with a vesicular uptake-storage mechanism for catecholamines. It is proposed that in infant rat there is a major projection from brain stem MA nuclei to the neocortex; this projection may exert a potent influence on the activity of neurons in layer IV.

A procedure for sampling specific portions of identified neurons for ultrastructural examination.

A procedure is described which facilitates the ultrastructural examination of selected regions of a neuron identified because it is Golgi-impregnated or HRP-filled. The procedure involves assigning a two-dimensional coordinate to every point on a drawing of the neuron by superimposing a grid over the drawing. A complete series of thick sections through the block containing the identified cells can then be related to each point on the drawn cell. A thick section which contains a region of interest is remounted on a plastic blank. From this new block one obtains thin sections for ultrastructural examination.

Horseradish peroxidase pellets implanted into infant neocortex: some technical considerations.

This study was undertaken to examine whether implanting pellets of horseradish peroxidase (HRP), rather than injecting an aqueous solution, would improve the sensitivity of the retrograde tracing method as applied to infant rat neocortex. From 1 to 10 pellets, each containing approximately 10 microgram of HRP, were implanted into somatosensory cortex of 6-day-old rats. Implantation of one pellet labeled 4 neuronal groups; 5 pellets, 37 groups. Higher doses of injected HRP (20--50 microgram and 200-400 microgram) are needed to label the same number of groups. Also, individual neurons of a group generally contain more granules/cell after pellets than following injections of much higher doses of HRP. The pellet implant technique offers a high degree of reproducibility and is technically simpler than injections. We conclude that HRP pellet implants offer advantages over injections in identifying potential afferents to immature neocortex.

Scalloping deformity of the corpus callosum following ventricular shunting.

PURPOSE: To describe six patients who underwent ventriculoperitoneal (V-P) shunting for hydrocephalus and developed scalloping deformity of the dorsal surface of the corpus callosum, and to evaluate the cause and frequency of this phenomenon. MATERIALS AND METHODS: MR images of 35 patients whose hydrocephalus was successfully corrected by V-P shunting were studied. To elucidate the possible anatomic basis for the scalloping deformity, gross examination of the corpus callosum relative to the adjacent anatomical structures was performed using autopsy specimens. RESULTS: Of the 35 patients who underwent successful V-P shunting, six (17%) developed a scalloping deformity of the corpus callosum. The deformity was noted exclusively in the body of the corpus callosum. This phenomenon was observed in both obstructive and communicating hydrocephalus regardless of the patient's age, but was particularly noted in patients with tectal tumors. CONCLUSION: The cause of this phenomenon may be a combination of long-standing hydrocephalus and normal pericallosal artery anatomy. Prolonged hydrocephalus softens the corpus callosum and the branches of the pericallosal arteries tether the corpus callosum to the overlying cingulate cortex at periodic intervals.

Posterior fossa glioblastoma multiforme: MR findings.

PURPOSE: To characterize the MR findings of glioblastoma multiforme in the posterior fossa. METHODS: MR studies of nine patients with surgically proved posterior fossa glioblastoma multiforme were retrospectively evaluated. MR characteristics studied included tumor location, signal intensity, enhancement pattern, and presence of intratumoral hemorrhage, as well as presence of secondary hydrocephalus or metastatic spread. RESULTS: The tumors were located in the median portion of the cerebellum or brain stem in eight cases. Six extended into the fourth ventricle. Hydrocephalus was seen in four cases. Six cases demonstrated decreased T1- and increased T2-weighted signal intensities. Three cases demonstrated mixed signal intensities suggesting intratumoral hemorrhage. All of the eight patients who received contrast showed moderate to marked heterogeneous ringlike enhancement suggesting intratumoral necrosis. Multicentric/multifocal lesions or extraaxial metastases were identified in three of the nine cases, and there was extracranial extension into the cervical region in one case. CONCLUSION: Glioblastoma multiforme is a rare tumor in the posterior fossa. Differentiating it from metastatic tumor or malignant astrocytoma was difficult. However, combination of heterogeneous and ringlike enhancement, midline location, poorly defined margin, tumoral hemorrhage, concomitant multicentric/multifocal lesions, and extraaxial or extracranial metastasis may be clues for the prospective diagnosis of glioblastoma multiforme.

Penetration and internalization of plasma proteins in the human spinal cord.

In animal studies, motoneurons take up plasma proteins including immunoglobulins at their terminals. These proteins are then transported back to cell bodies in the spinal cord. To determine if these processes also occur in humans, we localized several different plasma proteins in autopsied spinal cords from 13 patients without neurological disease. As in animals, plasma proteins are associated with vascular and pial structures. Motoneurons, particularly large cervical and lumbar motoneurons, frequently showed immunoreactivity within their cytoplasm to several plasma proteins. Motoneuron labeling was more consistent with antisera against plasma proteins of lower molecular weights such as IgG, IgA and transferrin, than with antisera against higher molecular weight proteins such as IgM and alpha-2-macroglobulin. Other large neurons without connections outside the blood-brain barrier such as those of Clarke's column also occasionally labeled with antisera against all plasma proteins tested. Our results are compatible with the concept that motoneurons take up and transport plasma proteins. These neurons can be distinguished from cells which internalized extravasated serum proteins before and after death. Uptake of pathogenic antibodies by motoneuron terminals may play a role in the pathogenesis of motoneuron disease.

Internalization of plasma proteins by cerebellar Purkinje cells.

Animal studies suggest that Purkinje cells internalize proteins from the blood and CSF. This process may relate to the pathogenesis of paraneoplastic cerebellar degeneration in patients with anti-Purkinje cell antibodies. To determine if human Purkinje cells may also internalize plasma proteins, cerebellar tissue was taken from routine autopsies of eight patients without neurologic or neoplastic disease. Several plasma proteins including IgG, IgA, IgM, transferrin, albumin and alpha-2-macroglobulin were detected by immunohistochemistry within the cytoplasm of Purkinje cells. Internalized proteins frequently filled the entire soma and major dendrites, sparing the nucleus. Vascular structures were also immunolabeled, while glia internalized plasma proteins differentially, with oligodendrocytes selectively internalizing transferrin. Purkinje cells were the most numerous and heavily labeled neuronal cell type in spite of their small numerical representation in the cerebellar neuronal population. Our results are compatible with previous animal studies, and suggest that internalization of specific antibodies could contribute to the pathogenesis of Purkinje cell loss in paraneoplastic cerebellar degeneration.