A novel thin section preparation and staining protocol to increase contrast and resolution of cell details for light microscopy.

We developed a novel sectioning and staining method to make high contrast, high resolution sections of plant tissue for light microscopy. Specimens of teosinte (Zea mays L., ssp. mexicana) root tips were fixed and embedded in Technovit 7100™ plastic resin. Thin sections, 1-2.5 µm, were cut and mounted on glass slides. The sections were either treated with RNase or not, then stained with 0.1% toluidine blue O and observed through ∞/0 objective lenses. For light microscopy, the enzyme staining procedure increased resolution and contrast. High magnification ∞/0 objective lenses produced high quality images for digital photography without using a coverslip or immersion oil. Our slide preparation and microscopic analysis were less labor intensive and more rapid than previous methods and enabled rapid and precise alignment of serial transverse sections for both tracking cell lineages and tissue measurements.

Changes in growth and structure of pea primary roots (Pisum sativum L. cv. Alaska) as a result of sudden flooding.

Pea (Pisum sativum L. cv. Alaska) primary roots were exposed to flooding after growth for 4 or 5 d at 25 degrees C under relatively dry conditions. Flooding after 4 d growth reduced, but did not stop, primary root growth, and cavities caused by degradation of central vascular cells were typically found from 10-60 mm from the tips. Flooding after 5 d stopped primary root growth and caused cell death in the tips, and vascular cavities formed that typically were 20-60 mm from the tips of the roots. Degradation of root tip cells in 5-day-roots was very rapid and began in the elongation zone and later in the apical zone. Root tips discolored, narrowed or curled before growth arrest. The mitotic indices of 5-day-root tips were suppressed by the flooding treatment. A few mitotic figures were observed in roots treated with flooding after 4 d growth. Affected cells had condensed nuclei, but cytoplasms appeared to be normal in the early stages of cell degradation. Later these cells became very vacuolated. The relationship of flooding to root growth, vascular cavity formation, and the morphology of pea primary roots is described with regard to the ability to resist flooding stress.

Regeneration of tissue following cavity formation in the vascular cylinders of Pisum sativum (Fabaceae) primary roots.

The reorganization of vascular cylinders of pea (Pisum sativum, cv. Alaska) primary roots following the formation of vascular cavities was examined by light and electron microscopy. Cavities usually began forming ~20 mm from the root tip and were continuous to ~90 mm from the tips in roots 150 mm long, where they began filling with specialized parenchyma cells (SP cells). SP cells were usually produced by enlargement of parenchymous cells of the primary xylem at cavity margins. Depending on the extent and shape of the cavity, they were also sometimes produced by primary phloem parenchyma and early derivatives of the vascular cambium. Enlargement and some divisions of SP cells continued until a cavity was completely filled by them. SP cells proceeded through a series of cytoplasmic changes as they developed. First the cytoplasmic layer became thicker and more electron dense than surrounding cells. As SP cells enlarged there was an increase in vesicular traffic and the cytoplasm became less electron dense. Ultimately the cytoplasm thinned further, organelles degenerated, and the tonoplast sometimes broke down. SP cells did not form secondary walls. X-ray microanalysis revealed that SP cells accumulated potassium and rubidium to the same degree as cortical and xylem parenchyma cells and to a greater degree than immature secondary and late-maturing tracheary elements.

Botulinum toxin injection for cervicogenic headache.

We report a 28-year-old woman with a 5-year history of cervicogenic headache following a whiplash injury. Her unilateral neck pain, if aggravated by exertion, would create a predictable sequence of events leading to a hemicephalgia. She proved medically refractory to usual therapies, but had a striking response to a single botulinum toxin Injection in her symptomatic trapezius muscle. Repeated Injections every 3 months have been required to maintain this benefit. The Implications of this observation are discussed.