Modification of AgNOR staining to reveal the nucleolus in thick sections specified for stereological and pathological assessments of brain tissue.

BACKGROUND: Various stains have been devised to reveal degenerative or reactive cell phenotypes, or the disintegrative and/or neuropathic lesions associated with Alzheimer's, Parkinson's, and Pick's diseases, Down's syndrome, or chemical toxicity. Utilization of silver staining has allowed researchers to elucidate neural pathways promoting a greater understanding of the functional connections between brain regions. All of these methods employing silver can be characterized as 'directed staining technologies'. NEW METHODS: The argyrophilic proteins (AgNOR) staining protocol was modified to stain nucleoli in thick sections prepared for stereological evaluation of brain tissue. Nucleoli appeared as black dots against a pale amber background. Tissue sections were counterstained with Toluidine Blue, or reduced-strength Tyrosine Hydroxylase immunohistochemistry to facilitate visualization of basic cellular morphology and regional nucleus identification. Here, we present a modified method for nucleolar staining in free-floating thick sections of brain embedded in a gelatin matrix. The modifications in our procedure include incubation in HCl to denature ('unravel') the DNA, a bleaching step to reduce non-specific background silver staining, and counterstaining with Toluidine Blue or reduced-strength tyrosine hydroxylase immunohistochemistry. COMPARISON WITH OLD METHODS: Prior to the development of immunohistochemistry, silver staining was used primarily to identify pathological profiles and trace axon pathways; however, in many cases, a combination of silver staining and immunohistochemistry are required to fully visualize pathomorphology. The mechanism of these stains requires the binding of silver ions to cellular components and the subsequent reduction of the ions to metallic silver. Dilutions of TH primary antibody were evaluated to maximize identification of neurons and the nucleolus amongst the soma and processes present in the thick section. The use of stereology as a tool to estimate cell number has become increasingly prevalent in neuroscience experiments. As requirements for the preparation of experimental tissue have been refined, researchers have begun to use thicker sections, between 40 to 80 microns, to increase the number of optical planes available for analysis. These thick sections require modified staining protocols to assure complete penetration of stains throughout the tissue section. CONCLUSIONS: This method is particularly useful in nucleolar identification for Stereology, and automated counting methods. Use of the nucleolus avoids some of the problems associated with use of the nucleus. The nucleolus is smaller than the nucleus and is less susceptible to transection during sectioning. It has a higher density than the nucleus and is easier to visualize. It is generally darker staining than the immunohistochemical reaction product that provides the identification marker for the cells to be counted. Examples of the method in several brain sections of the rat are shown, though the method has been also proven in other mammalian models.

The safety, tolerability, and pharmacokinetics of fosphenytoin after intramuscular and intravenous administration in neurosurgery patients.

STUDY OBJECTIVE: To evaluate the safety, tolerability, and pharmacokinetic profile of fosphenytoin, a water-soluble phenytoin prodrug, after intramuscular and intravenous administration. DESIGN: Open-label study of intramuscular administration, and double-blind, randomized study of intravenous administration. SETTING: Six and ten hospitals throughout the United States for the intramuscular and intravenous multicenter studies, respectively. PATIENTS: Neurosurgical patients who required anticonvulsant prophylaxis or treatment. INTERVENTIONS: In the intramuscular study, 118 patients received loading doses ranging from 480-1500 mg phenytoin equivalents (PE) and daily maintenance doses ranging from 130-1250 mg PE for 3-14 days. In the intravenous study, 88 patients received fosphenytoin and 28 received phenytoin sodium for 3-14 days. Mean +/- SD loading doses and maintenance doses of intravenous fosphenytoin and phenytoin were 1082 +/- 299 mg PE and 411 +/- 221 mg PE, and 1082 +/- 299 mg and 422 +/- 197 mg, respectively. Trough phenytoin concentrations were measured daily in all patients. MEASUREMENTS AND MAIN RESULTS: Intramuscular fosphenytoin was safe and well tolerated, with no irritation found for 99% of all injection site evaluations. Adverse events associated with the drug occurred in 9% of patients, commonly those typical of the parent drug. For intravenous treatment, the frequency of mild irritation at the infusion site was significantly lower in the fosphenytoin group (6%) than in the phenytoin group (25%, p < 0.05). Reductions in infusion rates were required in 17% and 36% of fosphenytoin and phenytoin recipients, respectively. No significant difference was observed relative to adverse events or seizure frequency between the groups. Trough plasma phenytoin concentrations were approximately 10 micrograms/ml or greater in patients receiving at least 3 days of intramuscular and intravenous fosphenytoin. Trough phenytoin concentrations were similar between patients receiving intravenous phenytoin and fosphenytoin on all study days. CONCLUSION: Fosphenytoin can be administered intramuscularly and intravenously in neurosurgical patients to achieve and maintain therapeutic phenytoin concentrations for up to 14 days. Both routes are safe and well tolerated. Intravenous fosphenytoin is significantly better tolerated than intravenous phenytoin sodium in this patient subset.

Embolism to the central nervous system from cardiac myxoma. Report of two cases.

Two cases of left atrial myxoma are reviewed, both presenting as embolic phenomena. Neither patient gave a history compatible with pre-existent cardiac dysfunction. Sudden collapse and subsequent right hemiplegia resulted in one patient when an embolus lodged in the left middle cerebral artery. The second patient presented with headache and transient visual obscuration in the left eye. She showed evidence of embolism to the central retinal artery, and particulate matter could be seen within the retinal arterioles. Attention is drown to the fact that echocardiography now constitutes a simple, noninvasive, and highly reliable method of making this diagnosis. The propensity for embolic tumor fragments to grow and invade cerebral arterial walls is discussed along with its possible neurosurgical significance.

Direct coupling of expanded bed adsorption with a downstream purification step.

In the course of developing a cost-effective, scaleable process for the purification of a recombinant protein from Chinese hamster ovary (CHO) suspension cell culture, we investigated direct capture of this molecule using expanded bed adsorption (EBA). EBA combines clarification, purification, and concentration of the product into a single step. The unclarified bioreactor material was directly applied to a STREAMLINE 25 column containing an affinity STREAMLINE adsorbent. This work focused on simplifying the EBA operations and minimizing the overall processing time by running the EBA column unidirectionally, eluting in the expanded bed mode, and coupling the EBA column directly with ion exchange or hydrophobic interaction chromatography. Unidirectional EBA was clearly a simpler unit operation and did not require the use of specialized equipment. The increase in the elution pool volume was insignificant, especially when the EBA column was eluted directly onto the downstream column. Scale-down was simple and could be automated. Coupling of unidirectional EBA with a downstream purification step reduced processing time, equipment requirements and cost.

Hepatic encephalopathy mimicking stroke. A case report.

This case demonstrates focal neurologic deficit mimicking stroke with underlying hepatic encephalopathy. Unilateral weakness in patients with hepatic encephalopathy has not been previously described in the English language literature. A 46-yr-old white woman was admitted to an acute care hospital for left shoulder manipulation, underwent general anesthesia and appeared to have had a right cerebrovascular accident. At transfer to the rehabilitation hospital, in addition to the left hemiparesis, there were inconsistencies in the neurologic examination and signs of cognitive impairment and liver failure. The patient's response to an intensive, multidisciplinary inpatient rehabilitation program along with treatment of the liver dysfunction led to resolution of left-sided weakness and flapping tremor with independence in ambulation and activities of daily living. Relevant literature is reviewed. A thorough history and physical examination with liver function assessment should always be performed in patients with cerebrovascular accident and unusual recovery.

Carbofuran toxicosis in cattle: case history and analytical method.

Thirteen head of crossbred Brahman cattle were exposed to carbofuran which had been mixed with rice to control blackbirds. Three animals died within 20 minutes after eating the contaminated grain, and the other 10 exhibited clinical signs associated with carbamate toxicosis. The 4 most severely affected animals were treated with a subtherapeutic dose of atropine sulfate; they did not respond and subsequently died. The remaining 6 recovered. Samples of rumen contents from the animals that died contained carbofuran concentrations ranging from 2 to 51 mg/kg.

Measurements of ultracold-neutron lifetimes in solid deuterium.

We present the first measurements of the survival time of ultracold neutrons (UCNs) in solid deuterium (SD2). This critical parameter provides a fundamental limitation to the effectiveness of superthermal UCN sources that utilize solid ortho-deuterium as the source material. These measurements are performed utilizing a SD2 source coupled to a spallation source of neutrons, providing a demonstration of UCN production in this geometry and permitting systematic studies of the influence of thermal up-scatter and contamination with para-deuterium on the UCN survival time.