An animal model of life-threatening hyperthermia during infancy.

A mathematical model of heat balance in human infants suggests that it may be possible for severe hyperthermia to develop if an infant is unable to remove his blankets in response to overheating (thermal entrapment). This hypothesis was tested in an animal model of weanling piglets. Ten piglets were warmed in a radiant heater to rectal temperature of 41 degrees C to simulate a fever. Animals in the experimental and control groups were removed from the heater and covered with ordinary infant blankets (to a thickness of approximately 3 cm). Endogenously produced heat caused the animals to warm to 42 degrees C. At this point, the control animals were uncovered. They rapidly cooled to normal body temperature. Animals in the experimental group remained covered until they expired from hyperthermia at 43.9 +/- 0.7 degrees C (SD) after 96 +/- 43 (SD) min. These data show that lethal hyperthermia may result from thermal entrapment. This finding may help clarify the role that hyperthermia may play in illnesses such as hemorrhagic shock and encephalopathy syndrome and some cases of sudden infant death syndrome.

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.

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.

Acetylcholine receptor density and acetylcholinesterase activity in skeletal muscle of rats following thermal injury.

Thermal injury causes systemic changes that result in altered sensitivity to many drugs including nondepolarizing muscle relaxants. In an effort to identify the mechanism(s) responsible for the resistance to nondepolarizing muscle relaxants, the density of acetylcholine receptors (AChR) and the activity of acetylcholinesterase (AChE) were determined in rats following a 30% total body surface thermal injury at a time when resistance to atracurium is maximum. AChR density in gastrocnemius and diaphragm was unchanged by thermal injury. Furthermore, the ratio of junctional to extrajunctional AChR in diaphragm was unaltered. Total AChE activity was unchanged in thermally injured rats compared with that in sham-injured animals. Separation of the molecular forms of AChE by sucrose gradient centrifugation also showed no changes in the relative proportions of these species. The mechanism of resistance to nondepolarizing muscle relaxants does not appear to be explained by changes in AChR number or changes in the activity of AChE.

Resistance to atracurium in thermally injured rats. The roles of time, activity, and pharmacodynamics.

Thermal injury induces resistance to nondepolarizing muscle relaxants in patients. Because the mechanism of the resistance is unknown, the authors have sought to establish thermally injured rats as a suitable model for subsequent detailed studies of mechanisms. Two hundred twenty-five- to 250-g rats sustained a 30% total body surface area thermal injury while anesthetized with pentobarbital. Another group had sham injury. Animal activity was monitored both by periods of direct observation and by use of activity cages. At 10, 20, 30, 40, 60, and 90 days after injury, rats were anesthetized and ventilated and the strength of contraction of their gastrocnemius produced by supramaximal stimulation of the sciatic nerve was measured before and after a bolus of atracurium (2.0 mg/kg) was administered. The plasma concentration required to diminish contraction to 50% of the preceding value (Cp50) was determined by atracurium infusion. Animals displayed the greatest resistance to atracurium at 40 days. The Cp50 value was also greatest at this time. The protein binding of atracurium was identical for both sham and injured groups. Activity for thermally injured resistant rats and for sham animals was not different. It appears that pharmacodynamic mechanisms are involved, and inactivity and disuse atrophy are not necessary in rats for development of resistance to nondepolarizing muscle relaxants after thermal injury.

Release of neuronotrophic factor from rabbit corneal epithelium during wound healing and nerve regeneration.

Epithelial neuronotropic factor (ENF) is secreted by cultured epithelial cells of rabbit cornea and conjunctiva, and is active in promoting survival and inducing neurite outgrowth of cultured trigeminal neurons. This study evaluated the relation of ENF to corneal nerve regeneration utilizing a model of heptanol-induced epithelial wounding. The organ culture technique was used to collect ENF from the intact corneal epithelium, and a neuronal bioassay was utilized to quantify ENF. The results revealed no change in ENF secretion either during initial wound closure or after 1 week, when the epithelium had regenerated. However, ENF secretion was elevated 2.4 times in 2 weeks after wounding. Morphometric analysis of corneal nerves stained by gold chloride impregnation showed that the first sign of regeneration of intraepithelial nerves was observed after 2 weeks, and the normal pattern of epithelial neural density was re-established after 3 weeks. However, the neural density was still subnormal (35-47% less than the control) in the wounded epithelium up to 4 weeks after wounding. Thus it appears that a surge in ENF secretion occurred after epithelial regeneration but before nerve regeneration. The results suggest that ENF may mediate corneal nerve regeneration.

Specificity of a neuronotrophic factor from rabbit corneal epithelial cultures.

Previous studies have demonstrated that of the three major cell types of rabbit cornea, only epithelial cells released a neuronotrophic factor that is active in both promoting survival and inducing neurite outgrowth of dissociated trigeminal neurons. In this study, the specificity of neuronotrophic factor production by selected epithelial cell types and the responsiveness of neurons from several different tissues to this trophic factor were determined. Cell cultures of various epithelia and dissociated neurons from several ganglia of neonatal and adult rabbits were utilized. In comparison with adult corneal epithelium, adult bulbar conjunctiva released a similar amount of trophic factor. Neonatal corneal epithelium released five times more, but adult retinal pigment epithelium and neonatal skin epithelium yielded less than a third and one-sixth respectively, and three cell lines (adult skin, kidney and neonatal lens epithelia) released only negligible amounts. Compared with its effect on neonatal trigeminal neurons, the trophic factor from adult corneal epithelium was similarly active on neonatal dorsal root neurons, but only half as effective on adult trigeminal neurons and less than one-tenth as effective on neonatal superior cervical neurons. The effect of nerve growth factor (from mouse submaxillary gland) on these neurons was tested for comparison. The results indicated that of the tissues studied, neuronotrophic factor was released in abundance only from corneal and conjunctival epithelia, and was active predominantly on trigeminal and dorsal root neurons (sensory neurons). This is consistent with the concept that the developmentally regulated release of trophic factor by target cells may be an underlying mechanism for neurogenesis and nerve regeneration.

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.

Electrical nerve location: numerical and electrophoretic comparison of insulated vs uninsulated needles.

We compared the electrical characteristics of insulated and uninsulated needles in two models that simulate use of a stimulator for nerve localization. With a digital computer, we solved for and graphed the contours of constant electric field strength, defining regions of simulated tissue in which a nerve would become depolarized for a particular stimulation current. We found that with an uninsulated needle, these regions extend proximally along the needle shaft with their widest dimension located slightly shallow to the tip, but with insulated needles, the regions are almost circular and are centered slightly deep to the needle tip. We confirmed these findings by electrophoresis of bromphenol blue dye in polyacrylamide gel. We also found that the necessary stimulator current is much more dependent on the depth of needle insertion with uninsulated needles than with insulated needles. We conclude that the electrical characteristics of insulated needles are more favorable for successful nerve block.

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.

Extravascular, extracellular water in rabbit lung using Na and sucrose indicators.

We compared the volume of the pulmonary extravascular, extracellular water space using sodium and sucrose indicators in 8 normal and 11 edematous rabbit lungs by steady-state techniques. The volume of the extravascular, extracellular space (EVECW), expressed in milliliters per gram dry lung, was determined from the volume of distribution of 24Na, [3H]sucrose, or total sodium in 1) supernatant solutions obtained by centrifugation of homogenized lung or 2) intact lung fragments. There was no significant difference in EVECW measured in normal lungs [24Na 1.88 +/- (SD) 0.31; [3H]sucrose 1.84 +/- (SD) 0.27] or edematous lungs [24Na 4.78 +/- (SD) 1.44; [3H]sucrose 4.41 +/- (SD) 1.33] when comparing supernate solutions of homogenized lungs. Similarly, there was no significant difference between the volumes of distribution (EVECW) of these two isotopes when measured in intact fragments of normal lung [24Na 2.08 +/- (SD) 0.28; [3H]sucrose 1.68 +/- (SD) 0.37]. The volume of EVECW measured in supernates was less than in fragments with both isotopes. Washing the centrifuged tissue pellet and adding the wash activity to the initial supernate activity yielded results that were equal to the fragment technique. The failure to detect significant differences between 24Na and [3H]sucrose spaces in the lung suggests that the contribution on intracellular sodium to the measurement of lung water is small and relatively insignificant, at least within the confines of this experiment.

Action of a trophic factor(s) from rabbit corneal epithelial culture on dissociated trigeminal neurons.

Cultured corneal epithelial cells release a factor(s) that stimulates trigeminal neurons to form neurites in vitro. To characterize this trophic effect, conditioned media (serum free, supplemented) from cultures for corneal epithelium, stromal fibroblasts, and endothelium were studied further. Only epithelial conditioned medium (PCM) prolonged neuronal survival and induced neurite outgrowth. This trophic influence peaked after 2 to 3 days and gradually declined thereafter during a week when the medium was not renewed. Using a bioassay to score the percentage of initially viable neurons that extended neurites, it was found that the trophic effect of PCM was proportional to the conditioned medium concentration and to the cell density of the epithelial culture used for the conditioning. Maximum activity in PCM was correlated with confluency of the epithelial culture. Experiments using antiserum to nerve growth factor (NGF) and purified antibody to cold-insoluble globulin (CIG) indicated that the tropic effect of PCM was not derived from NGF or CIG. The trophic activity of PCM was abolished totally by heat or trypsin treatment but was not affected by collagenase. Although a fraction of the trophic activity was associated with the substratum after adsorption of PCM, this and other evidence did not suggest that the primary action of PCM was to enhance neuronal adhesion.

Differential uptake of horseradish peroxidase isoenzymes by cultured neuroblastoma cells.

The endocytotic uptake and intracellular decay of horseradish peroxidase isoenzymes C and A by cultured mouse neuroblastoma cells were analyzed quantitatively by a direct spectrophotometric assay. At concentrations below 1 mg/ml, the rate of uptake of the isoenzyme C was more than three times as much as the isoenzyme A. This differential uptake suggests that previous claims of horseradish peroxidase being endocytosed only in the nonselective fluid phase are oversimplified. The implication of this selectivity in the biological significance of retrograde axonal transport of proteins by neuronal systems is discussed.

Preparation and retrograde axonal transport of an antiviral drug/horseradish peroxidase conjugate.

Recurrent Herpes simplex virus infections of the cornea are currently treated with antiviral drugs administered locally during periods of active infection. We have examined the feasibility of an alternate approach to treating recurrent infection, that of delivering an antiviral drug by axonal transport to cell somata in the trigeminal ganglion where the latent virus is thought to reside. We have coupled an antiviral drug to horseradish peroxidase (HRP), a protein which is readily transported retrogradely in corneal sensory axons to ganglion cell somata in the trigeminal ganglion. The antiviral drug 5-iodo-5'-amino-2',5'-dideoxyuridine (AIDU) was synthesized with 125I and coupled to horseradish peroxidase by Schiff base formation between the 5' amino group and aldehydes generated on the carbohydrate of the HRP following short periodate oxidation. The useful ratio of AIDU:HRP was 2-9; higher ratios resulted in an insoluble product. Sodium dodecylsulfate (SDS) acrylamide gel electrophoresis of the drug-protein conjugate revealed considerable aggregation and the isoelectric point of HRP was changed from 8.1 to 4.4-5.8 following the coupling procedure. Despite evidence that the protein conformation was considerably altered, the specific enzymatic activity of the final product was 58% of native HRP and the drug-protein conjugate was still strongly transported retrogradely. Retrograde transport of this conjugate was demonstrated by autoraiography of the trigeminal ganglion neurons 24 h after corneal injection of the [125I]AIDU/HRP.

Endocytosis and compartmentation of peroxidases and cationized ferritin in neuroblastoma cells.

To investigate further the selectivity of retrograde axonal transport of horseradish peroxidase (HRP) isoenzymes previously observed in the rat central visual pathways, cultured mouse neuroblastoma cells (clone N18) were examined for selectivity of endocytosis of peroxidases and cationized ferritin in vitro. Differentiating N18 cells were incubated with proteins in various timing and pulse-chase experiments, and examined for the ultrastructural localization of endocytosed protein using cytochemical techniques. Semi-quantitative morphometry was performed on some of the samples. Major findings were as follows. (1) The protein was internalized into vesicles (coated and uncoated), short tubules and occasional small cup-shaped bodies within the first few minutes, and transferred via tubules and uncoated vesicles to secondary lysosome-like organelles (vacuoles, multivesicular bodies and dense bodies) from 5 to 15 min, with dense bodies representing the final site of protein accumulation. All the proteins tested were endocytosed through the same pathways in the soma and neurite of the cell. (2) There was no indication of a net orthograde or retrograde neuritic transport proteins. (3) There were induced increases in both vesicle and tubule formation as a result of protein endocytosis, with HRP isoenzyme C showing the greatest effect. (4) The apparent rate of internalization of HRP isoenzyme C into vesicles during the initial 5 min was significantly greater than for other peroxidases amd cationized ferritim. (5) Relatively more tubules than vesicles were involved in the uptake of protein as endocytosis was prolonged from 5 min to 8 h, with HRP isoenzyme C showing the largest effect. (6) There were indications of preferential compartmentation of endocytosed protein into certain organelles.

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

The 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 Abeta and Aalpha 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.