Central nervous system inflammation and neurological disease in transgenic mice expressing the CC chemokine CCL21 in oligodendrocytes.

To study the biological role of the chemokine ligands CCL19 and CCL21, we generated transgenic mice expressing either gene in oligodendrocytes of the CNS. While all transgenic mice expressing CCL19 in the CNS developed normally, most (18 of 26) of the CCL21 founder mice developed a neurological disease that was characterized by loss of landing reflex, tremor, and ataxia. These neurological signs were observed as early as postnatal day 9 and were associated with weight loss and death during the first 4 wk of life. Microscopic examination of the brain and spinal cord of CCL21 transgenic mice revealed scattered leukocytic infiltrates that consisted primarily of neutrophils and eosinophils. Additional findings included hypomyelination, spongiform myelinopathy with evidence of myelin breakdown, and reactive gliosis. Thus, ectopic expression of the CC chemokine CCL21, but not CCL19, induced a significant inflammatory response in the CNS. However, neither chemokine was sufficient to recruit lymphocytes into the CNS. These observations are in striking contrast to the reported activities of these molecules in vitro and may indicate specific requirements for their biological activity in vivo.

Vacuolation in renal tubular epithelium of Cd-1 mice: an incidental finding.

Prominent cytoplasmic vacuoles were observed in renal tubular epithelial cells of the outer medulla in several kidneys from test article-dosed mice (Crl:CD-1 (ICR)BR VAF/PLUS) during routine light microscopic (LM) examination. Because the vacuolar change was detected infrequently and was not found in any control mice from that study, it was not clear whether the vacuolation represented a drug-induced change. To address this question, kidney sections from mice from multiple unrelated studies were examined by LM for similar vacuolar changes. Vacuolation was seen by LM in 2.3% of the control and 2.8% of the test article-dosed mice. Transmission electron microscopy (TEM) was also performed on kidneys with prominent light microscopic vacuoles in 5 control mice and 2 test article-dosed mice to further characterize the vacuoles. Ultrastructurally, the vacuoles contained fibrillar and finely stipled granular material or membranous whorls. Kidneys from control mice lacking light microscopic evidence of vacuolation had smaller vacuoles containing similar material when examined by TEM. Because vacuoles were present in both control mice and test article-dosed mice, it was concluded that the vacuoles were incidental and unrelated to compound administration. These studies also demonstrated that vacuoles can be expected to be observed by LM examination in 2-3% of Crl:CD-1 (ICR)BR VAF/PLUS, mice.

Computed three-dimensional reconstruction of median-eminence capillary modules: image alignment and correlation.

Image alignment is an absolute requirement for three-dimensional (3-D) reconstruction from serial sections, and Fourier correlation is the most powerful way to compute alignments. The rotational and translational components of misalignment can be corrected by an iterative correlation procedure, but for images having significant differences, alignment can fail with a likelihood proportional to the extent of the differences. We found that translational correction was determined much more reliably when low-pass filters were applied to the product transforms from which the correlations were calculated. Rotational corrections based on polar analyses of the auto-correlations of the images instead of on the images directly contributed to more accurate alignments. These methods were used to generate 3-D reconstructions of brain capillary modules from serial-section mosaics of digitized transmission electron micrographs.

Computed alignment of dissimilar images for three-dimensional reconstructions.

Three-dimensional reconstructions from serial section images require the accurate registration of those images. Image correlation is the most powerful computed alignment method and its performance on identical images, or parts thereof, has been thoroughly studied. Correlation alignments of complex, dissimilar images can fail, however, with a likelihood proportional to the magnitude of the differences. We report that alignments can be computed more reliably and more accurately (higher-valued correlation coefficients) by the combined use of lowpass-filtered product transforms (from which the correlation functions are formed), autocorrelation correction of rotational misalignment, and covariance correction of translation misalignment. A simple rule is proposed for the lowpass filter cutoff radius depending on measures of the images' differences. These methods are demonstrated with a reconstruction of a capillary loop in the median eminence of the hypothalamus.

Three-dimensional reconstruction of median eminence microvascular modules.

To test the hypothesis that the median eminence microvasculature has a direct regulatory role in the hormonal communication between the brain and the pituitary gland, it is necessary to determine whether the physical means for such control (e.g. smooth muscle sphincters strategically located in the capillary plexus) actually exists. Our approach is to search for such structures in transmission electron micrographs of thin serial sections of the median eminence. The complexity of these images and the anticipated need to include large numbers of them in the study led us to consider computer reconstruction for this problem. We report here the successful three-dimensional reconstruction of capillary modules using digital image processing techniques for capillary feature detection/extraction, for construction of montages (mosaics) of overlapping images of the same section, and for automatic image registration by two independent methods without the use of fiducial marks. These tasks have been performed manually in nearly all the published neurobiological reconstructions; here they are performed by programs using only the mathematical properties of the images. Methods like those described here provide the only practical means for executing large scale reconstructions and gaining significant new information about the regulation of blood flow in this region of the brain.

Resistance vessels supplying the median eminence of the rabbit, rat, and cat.

The ultrastructure of arterioles supplying the median eminence of eight rats, eight rabbits, and two cats was studied after vascular perfusion with phosphate buffered aldehyde fixatives. There were terminal arterioles with a lumen diameter of 50-70 micron within the pars tuberalis. Smaller arterioles (precapillary sphincters and metarterioles) with a lumen diameter of 15-20 micron were present on the surface of the median eminence. Arterioles were not observed to penetrate the neuropil but were seen to supply the external capillary plexus of the median eminence. Direct innervation of arterioles supplying the median eminence was not present and hence regulation of median eminence blood flow by peripheral sympathetic mechanisms appears unlikely. Resistance vessels were found to be closely related to axon terminals on the surface of the median eminence and to fenestrated capillaries of the external plexus. In addition, the endothelial cells of arterioles were characterized by the presence of pits and vesicles which may play a role in transendothelial transport. These findings suggest two mechanisms by which blood flow into the median eminence can be regulated: (a) by central catecholaminergic systems terminating in the median eminence and (b) by catecholamine secretions from the adrenal medulla.

Neurohemal contact in the internal zone of the rabbit median eminence.

The concept of neurosecretion as the mechanism by which neural control of adenohypophyseal function is accomplished was based on the observation that long capillary loops penetrate deeply into the supraopticohypophyseal tract as it passes through the median eminence internal zone. However, neural contact upon these capillary loops has not been demonstrated in the mammalian median eminence. The present transmission electron microscopic investigation of the rabbit median eminence demonstrates neurohemal contact in the median eminence internal zone. Axons containing small lucent vesicles 53.3 +/- 3.28 nm in diameter (mean +/- SEM) or small lucent and large granular vesicles with a mean diameter of 122.4 (+/- 3.28 nm) in their terminals make neurohemal contact with capillary loops in the internal zone and form a cuff about them. These terminals resemble terminals found in the external zone. Intravenous injection of the false neurotransmitter 5-hydroxydopamine (5-OH-DA) renders small lucent vesicles granular in both the external and internal zone. The effect of 5-OH-DA injection is abolished by concurrent reserpine administration. Whereas large granular vesicles in many terminals become lucent after reserpine administration, in others they remained electron dense. Viewed in the light of previous studies our findings suggest that the internal plexus arises from the external plexus and invaginates the neuropil carrying connective tissue and parvicellular axon terminals of aminergic and peptidergic systems from the external zone into the internal zone, that some elements making neurohemal contact with long capillary loops are terminals of the noradrenergic reticular infundibular tract arising outside the hypothalamus in the brainstem, and that long capillary loops form a system of repeating microvascular modules which markedly increase the surface available for neurohemal contact.