Richard H. Haschke

Neurotoxicity of Intrathecal Local Anesthetics in Rabbits

The authors developed a new method of intrathecal local anesthetic injection in rabbits in order to study the relationship between anesthetic concentration and impaired neurologic function. They found that none of the local anesthetics studied produced persistent neurologic damage in concentrations used clinically. However, lidocaine and tetracaine can be prepared in high concentrations (far exceeding those clinically used) that will produce extensive irreversible neurologic injury and histologic changes. This was also true for sodium bisulfite, an antioxidant used in a number of commercially prepared local anesthetic solutions. Pure solutions of relatively insoluble local anesthetics (bupivacaine and 2-chloroprocaine) failed to produce comparable neurologic or neuropathologic changes when tested at concentrations up to their solubility limits. Extensive neurologic impairment was not necessarily accompanied by equally extensive lesions in the spinal cord and nerve roots.

Calcium-related properties of horseradish peroxidase☆

Abstract Horseradish peroxidase has been shown to be a metalloprotein in which calcium contributes to the structural stability of the protein. Isoenzyme C and A contain 2.0 and 1.4 moles calcium/mole enzyme, respectively, which can be removed by treatment with guanidine hydrochloride and EDTA. Calcium-free isoenzyme C, but not isoenzyme A, reconstitutes upon addition of calcium and regains enzymatic activity. Free calcium readily exchanges with isoenzyme C, but only to a small extent with isoenzyme A. In addition the role of calcium in maintaining molecular conformation is evidenced by the effects of calcium removal from the isoenzyme C on the thermal stability of the protein.

Spinal needle determinants of rate of transdural fluid leak.

Using a new in vitro model and samples of human dura, a number of factors related to spinal needle design and use were examined with respect to their effects on the rate of transdural fluid leak. These included needle size, bevel design, bevel orientation, and angle of approach. Using 25-gauge Quincke needles, a 30 degree approach caused a rate of leak across the dura significantly less than those following 60 degree and 90 degree approaches. A significant increase in leak rate was found with 22-gauge Quincke needles when the bevels were oriented so as to be perpendicular rather than parallel to the long axis of the dura. Also, 22-gauge Whitacre needles caused significantly less leak than did 22-gauge Quincke needles, and 25-gauge Quincke needles produced significantly less leak than 22-gauge Quincke needles. If human dura behaves in vivo as it does in this in vitro model, it would be advantageous to perform lumbar puncture using oblique approaches and small needles with conical tips.

Local Anesthetic Inhibition of Phagocytosis and Metabolism of Human Leukocytes

The effects of lidocaine on latex-particle phagocytosis and the metabolism of normal human leukocytes in citro were determined. A dose-dependent inhibition of nitroblue tetrazolium dye reduction which ranged from 8 per cent with less than 0.05 per cent lidocaine to 50 per cent with more than 0.45 per cent lidocaine was observed. Latex-stimulated leukocytic oxygen consumption was 59 per cent of control with 0.1 per cent lidocaine and 18 per cent of control with 0.5 per cent lidocaine. Lidocaine in concentrations of 0.1 per cent or more caused more than 50 per cent reduction in the number of cells engulfing latex. Leukocytic viability was impaired when cells were incubated with 0.5 per cent lidocaine for more than 30 minutes. Lidocaine therefore interferes with normal function of cells fundamental in defense against infection.

Effect of Thermal Injury on the Pharmacokinetics and Pharmacodynamics of Atracurium in Humans

Thermal injury causes resistance to many nondepolarizing muscle relaxants including d-tubocurarine, metocurine, pancuronium, and atracurium. To evaluate the role of pharmacokinetics and pharmacodynamics in this phenomenon, the disposition and effect of atracurium (0.5 mg/kg iv) were studied in thermally injured patients (5 males, 16-43 yr) in comparison with that in nonburned control patients (3 males, 1 female, 24-53 yr). The decline of plasma atracurium concentration with time was biexponential in both groups of patients. There were no significant differences in the mean value of any pharmacokinetic parameter (clearance, V1, V beta, alpha and beta half-lives). The time course of effect was also similar, although the maximum twitch depression was significantly smaller (66.1% vs. 100% maximal twitch depression) and time to recover to 50% of maximal twitch depression was significantly shorter (14.2 vs. 52 min) in thermally injured patients. Patients with thermal injury had an EC50 (plasma concentration of atracurium required for 50% of the maximum possible response) 3.4 times that of control patients. Plasma-free fraction of atracurium in the thermally injured patients was 75% that in controls, and free EC50 (the product of free fraction and EC50) of the thermally injured group was 2.7 times that of controls. The results of this study confirm a pharmacodynamic mechanism for the majority of resistance to atracurium, with a diminished free fraction in plasma also contributing to this effect.

Epithelial-stromal interactions: Specific stimulation of corneal epithelial cell growth in vitro by a factor(s) from cultured stromal fibroblasts

By using a recently modified method of isolating and culturing rabbit corneal cells, this study investigated the presence of a diffusible substance(s) in stromal fibroblast conditioned medium that stimulated the growth of cultured corneal epithelial cells. The growth stimulation involved initiation of DNA synthesis (assayed by [3H]-thymidine incorporation) and enhanced cell proliferation (quantified by cell counting). Among the three corneal cell types, only fibroblasts (rabbit and human) released the stimulatory substance, which acted only on epithelial cells. The effect of this stromal fibroblast factor (SFF) was observed after an exposure period of less than 16 hr and persisted as long as it was present. Its action was concentration-dependent and was not a result of improvement in the survival of epithelial cells during culture. Both sparse and confluent epithelial cultures were susceptible to SFF. The release of SFF was correlated with the number of fibroblasts in the culture and appeared to be sensitive to the growth condition of the cells. Both the release and action of SFF did not depend on the presence of serum in the culture medium. The factor was heat resistant and insensitive to proteolytic enzymes. From ultrafiltration studies, the size of SFF was estimated to be in the approximate range of 50-1000 daltons. By direct comparison of the stimulatory effect with other previously studied growth promoting agents, it was concluded that SFF was not epidermal growth factor, fibroblast growth factor, putrescine, cyclic AMP, hydrocortisone or acetate. The implication of SFF in the regulation of epithelial growth by endogenous, intercellular mechanisms is discussed.

Isolation and culture of corneal cells and their interactions with dissociated trigeminal neurons

The three cell types of rabbit cornea (epithelium, stromal fibroblasts and endothelium) were isolated by an improved method using both microdissection and selective enzyme treatment. This technique reproducibly resulted in an almost total recovery of each cell type from a given cornea. When maintained in culture, the three cell types showed different morphologic characteristics, each resembling the in vivo counterpart. The epithelial culture consisted of both attached and floating cells. The attached cells located at the marginal area of a colony were irregular in shape and possessed pseudopodia, while those in the confluent area were polygonal. Floating cells were typically vacuolated, curve-shaped and joined in groups of 2-4 cells as a spherical body enclosing a lucent interior. Comparison of mitotic rates, ultrastructure, keratin levels and other cytologic evidence suggested that the attached cells may correspond to the basal cells and less differentiated wing cells, while the floating cells may be analogous to the more differentiated wing cells and superficial cells. Neurons dissociated from neonatal rabbit trigeminal (Gasserian) ganglia were plated into multiwells partially covered with a given corneal cell type. The percentages of viable and neurite-bearing neurons were evaluated on the first three days. When neurons were grown in contact with each of the corneal cell types, neurites were extended in every case. However, when neurons were not in contact with the corneal cells in the coculture, only epithelial cells permitted neurite outgrowth. The data suggested two types of cellular interactions between corneal cells and sensory neurons, one of which may be the specific release of a neuronotrophic factor by epithelial cells. This culture system represents the first step towards developing an in vitro model for studying various cornea-trigeminal interactions.

EFFECTS OF LIDOCAINE ON RABBIT BRAIN MICROTUBULAR PROTEIN

Microtubule repolymerization from a crude supernatant fraction prepared from rabbit brain was followed quantitatively by viscometry and electron microscopy. Lidocaine inhibits this repolymerization in a dose‐dependent fashion and the effect is completely reversible upon removal of the lidocaine by dialysis. Direct counting of microtubules by electron microscopy indicates that the local anesthetic decreases the number of tubules without significantly affecting their length. An association constant of lidocaine for tubulin is estimated at 7.5 mm. Procaine and etidocaine were also found to affect the polymerization of microtubules with results similar to those found with lidocaine.

Features of foreign proteins affecting their retrograde transport in axons of the visual system

SummaryThe retrograde axoplasmic transport of foreign proteins in the rat visual system shows certain specificities. The molecular features of these proteins which may underlie their entry into the retrograde phase have been examined using biochemical and morphologic techniques. Of the isoenzymes of horseradish peroxidase (HRP), the basic isoenzyme C is strongly transported, while the acidic isoenzymes Aβ and Aα are transported weakly and not at all, respectively. Decreasing the isoelectric point (pI) of isoenzyme C from 8.2 to 4.4 decreases its transport, but a basic pI is not the sole requisite for transportability since two other basic peroxidases (turnip isoenzyme P7 and lactoperoxidase) are not transported in retrograde. The sugar component as a whole of isoenzyme C does not appear to be required for determining transport. Isoenzyme C and the other proteins which are transported enter multivesicular bodies in axons and axon terminals, as well as synaptic and coated vesicles and fine tubules in axon terminals. The non-transported proteins enter only the vesicular organelles thought to be involved in neurotransmitter recycling in axon terminals and do not enter multivesicular bodies. Thus the two systems of axonal membraneous compartments involved in local synaptic recycling versus the retrograde phase of transport do not show the same specificity of uptake of extracellular tracers and can be dissociated by the experimental use of these peroxidases.

An automated assay for glycogen phosphorylase

Abstract An automated assay of glycogen phosphorylase based on the method of Hedrick and Fischer (6) was developed using the Technicon Auto-Analyzer system. Since a protein precipitate is formed when measuring the product (inorganic phosphate), a dialysis step is commonly employed. Dialysis is unnecessary because SDS is used to prevent any protein precipitation. The method, which can measure any enzyme releasing orthophosphate, has a smaller standard error than the test performed by hand.