Nearly fatal muscle carnitine deficiency with full recovery after replacement therapy.

A 23-year-old woman became quadriplegic and respirator-dependent after 18 years of weakness and rhabdomyolysis. Her muscle tissue and that of a deceased sister contained lipid-laden fibers. Treatment with D,L-carnitine 4 grams per day was followed by a dramatic improvement within 10 days. Muscle function was normal at 8 months and has remained so during 3 subsequent years of L-carnitine 3 grams per day. Pretreatment muscle biopsy had documented low levels of free carnitine and short-chain acylcarnitine compounds. Carnitine palmityltransferase was slightly elevated. The asymptomatic parents had low-normal muscle carnitine levels, slight increase in muscle fiber lipid droplets, osmiophilic lipid-laden Schwann's cell vacuoles, and myelin lamellae with different periodicities.

Modulation of endothelin-1 production by a pulmonary epithelial cell line. I. Regulation by glucocorticoids.

Endothelin-1 (ET-1) is one of the most potent bronchoconstrictor agents yet described. Bronchial epithelial cells of asthmatic patients in vivo express preproET-1 and in vitro release high amounts of ET-1. Healthy and chronic bronchitic controls do not express preproET-1 or release ET-1. Interleukin-2 (IL-2) and other cytokines up-regulate the in vitro ET-1 release in guinea pig airway epithelial cells. We explored whether two glucocorticoids, dexamethasone (Dex) and triamcinolone acetonide (TA), inhibit the synthesis and release of ET-1 by A549 cells, a transformed human pulmonary epithelial cell line, since ET-1 may have a basic role in the pathogenesis of asthma. Cells were grown to confluence in RPMI 1640 plus 10% fetal bovine serum (FBS). Cells were then cultured for 3 days without serum to obtain ET-1 basal levels. The effects of 10% FBS, IL-2 (10 U/mL), Dex, TA or mifepristone, a steroid antagonist (1, 10 or 100 nM), were evaluated on ET-1 as measured by radioimmunoassay (RIA). ET-1 production increased from 57.6 +/- 5 pg/mg cell protein at 6 hr to 170 +/- 9 pg/mg cell protein at 72 hr in control cultures. Ten percent FBS increased ET-1 production from 58.7 +/- 9.6 to 399 +/- 14.5 pg/mg cell protein. IL-2 significantly increased ET-1 from 100.7 +/- 6.1 to 144 +/- 6.7 at 24 hr and from 170 +/- 9 to 207.7 +/- 24 at 72 hr. Dex and TA (10 and 100 nM) at 24-72 hr decreased ET-1 under basal conditions. Both drugs (only at 100 nM) decreased ET-1 production in 10% FBS- and IL-2-stimulated cells. Mifepristone (10 and 100 nM) reversed the decreased production of ET-1 induced by Dex (100 nM) at 24-72 hr. Northern blot analysis showed that Dex (100 nM) decreased the expression of ET-1 mRNA at 6 and 24 hr, but that mifepristone (100 nM) reversed this effect in cells cultured with Dex. In conclusion, Dex and TA down-regulate the synthesis and production of ET-1 by this human pulmonary epithelial cell line under basal or stimulated conditions, and these effects are reversed by mifepristone. These findings suggest a novel mechanism of glucocorticoid effect during the treatment of asthma.

Low-level exposure to methylmercury modifies muscarinic cholinergic receptor binding characteristics in rat brain and lymphocytes: physiologic implications and new opportunities in biologic monitoring.

Methylmercury (MeHg) affects several parameters of cholinergic function. These alterations are thought to play a role in MeHg neurotoxicity. In vitro experiments have indicated that MeHg acts as a strong competitive inhibitor of radioligand binding to muscarinic cholinergic receptors (mAChRs) in rat brain. Furthermore, rat brain mAChRs share several pharmacologic characteristics of similar receptors present on lymphocytes. Using the muscarinic antagonist [(3)H]quinuclidinyl benzilate (QNB) to label receptors, we investigated the in vivo interactions of MeHg with rat brain mAChRs. We also investigated whether MeHg-induced central mAChR changes are reflected by similar alterations in splenic lymphocytes. Exposure to low doses of MeHg--0.5 or 2 mg/kg/day in drinking water--for 16 days significantly increased (20-44% of control) mAChRs density (B(max)) in the hippocampus and cerebellum without affecting receptor affinity (K(d)). The effect of MeHg did not occur immediately; it was not apparent until 2 weeks after the termination of treatment. No significant changes in [(3)H]QNB binding were observed in the cerebral cortex. In splenic lymphocytes, mAChR density was remarkably increased (95-198% of control) by day 14 of MeHg exposure and remained enhanced 14 days after the cessation of treatment. These results suggest up-regulation of mAChRs in selected brain regions (hippocampus and cerebellum) after prolonged low-level ingestion of MeHg in rats. These cerebral effects are delayed in onset and are preceded by a marked increase in density of mAChRs on lymphocytes. In chronic MeHg exposure, peripheral lymphocytes may represent a sensitive target for the interaction of MeHg with mAChRs and, therefore, may be predictive indicators of later adaptive response involving cerebral mAChRs. Additionally, the effect of MeHg on lymphocyte mAChRs in vivo indicates that this receptor system should be investigated further as a possible target for MeHg immunotoxicity.

A mouse model for the study of blood-brain barrier permeability.

This article describes a C57BL/6 mouse model for the investigation of blood-brain barrier (BBB) alteration. Osmotic modification of BBB was achieved by infusion of 1.6 M arabinose solution into the internal carotid artery with or without occlusion of the external carotid artery. BBB alteration was measured by infusing 2% Evans blue dye. Only 1.6 M arabinose-treated animals but not 0.9% NaCl controls displayed prominent ipsilateral staining of frontal and temporal lobes. Light blue staining occurred in animals sacrificed within 10 min after injection. Prominent staining occurred in animals sacrificed 1-6 hours later. Identically treated animals were maintained for up to 6 months without signs of systemic or neurological dysfunction. This model may permit study of the effects of biological response modifiers (BRMs) upon the central nervous system (CNS) in healthy and diseased mice.

Nerve and muscle damage after experimental thrombosis of large artery. Electrophysiology and morphology.

The effect of the terminal aorta thrombosis on the spinal cord and hind limb nerves and muscles morphology, and the sciatic-tibial motor nerve conduction was studied in cats. The effect of the iliac and femoral artery thrombosis on nerve morphology and conduction was also examined. Aortic thrombosis usually caused severe nerve and muscle lesions while spinal cord was spared. Nerve and muscle damage was strikingly more extensive and severe after aortic thrombosis than ligation. Nerve damage was also seen after the iliac or femoral artery thrombosis but not after ligation of these arteries. The tibial and peroneal nerve segments at the calf level were most vulnerable to ischemic damage. The nerve conduction studies (NCS) localized nerve lesions and indicated severity of the morphologic changes. The nerve conduction changes after arterial thrombosis reached a nadir at more variable time than in other experimental models of peripheral nerve ischemia. The markedly delayed development of maximal nerve dysfunction in some cases, if confirmed in humans, may present a rationale for aggressive medical or surgical intervention even several hours after acute arterial thrombosis.

Ameliorating effects of hyperbaric oxygenation on experimental allergic encephalomyelitis.

The effect of hyperbaric oxygenation (OHP) on survival and quality of survival of guinea pigs afflicted with experimental allergic encephalomyelitis (EAE) was investigated. EAE was induced in Hartley and Strain 13 animals by intradermal injections of whole guinea pig spinal cord in complete Freund's adjuvant. The inoculated animals were divided into control and treatment groups; the treated animals received OHP in a variety of treatment schedules. Clinical signs of EAE were quantitated and mean survival times were measured. When Hartley animals were exposed to 100% O2 at 2.5 atmospheres absolute (ATA) for 2 hr/day from 5--19 days postinoculation, the mean survival time (+/- SE) was 19.1 +/- 1.6 days relative to 15.7 +/- 0.7 days in the control (p less than 0.050). When Strain 13 guinea pigs were treated with 100% O2 at 2ATA for 4 hr/day on 5--16 days, the mean survival time was 21.6 +/- 0.6 days compared to 16.0 +/- 0.4 days for the control (p less than 0.001). Clinical sign measurements demonstrated that the onset of EAE in the treated animals of both strains occurred between 4--6 days after these signs became detectable in control animals. These results suggest that OHP therapy can ameliorate EAE in afflicted guinea pigs.

Cerebrospinal fluid lactate and electrolyte levels following experimental spinal cord injury.

Cerebrospinal fluid (CSF) lactate, sodium (Na+), potassium (K+), calcium (Ca++), magnesium (Mg++), and chloride (C1-) levels were determined for 17 to 21 days following experimental spinal cord compression in cats. Laminectomies were performed at L-2 under general anesthesia with aseptic techniques. Paraplegia was produced by applying a 170-gm weight transdurally for 5 minutes. Significant increases in CSF lactate levels were observed on the first through ninth days post injury with peak levels (50% above normal) occurring at Day 5. The only significant postinjury CSF electrolyte changes were elevation in Ca++ concentration on Days 3, 9, 11, 13, and 15, elevation in K+ concentration on Days 9 and 11 and decline in C1- levels on the first day. The CSF K+ increase probably reflected cellular loss of K+ from damaged tissue whereas the Ca++ rise may have resulted from increased CSF protein levels. The prolonged elevation of CSF lactate indicates that tissue hypoxia plays a role in spinal cord compression paralysis, and that there is a continuing hypoxia of metabolically active spinal cord tissue for several days post injury.

Modulation of glioma cell growth and 5-lipoxygenase expression by interferon.

The effect of mouse interferon-alpha/beta (MuIFN-alpha/beta on growth/viability, cell cycle regulation, 5-lipoxygenase (5-LO) protein expression, leukotriene B4 (LTB4) biosynthesis and glial fibrillary acidic protein (GFAP) expression of mouse glioma (G-26) cells in vitro was studied. The G-26 cells were treated with 800 IU/ml of MuIFN-alpha/beta for 1, 2, 3 and 4 days. The growth and viability of glioma cells was evaluated by [3H]-thymidine incorporation and MTT (3(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazoliumbromi de) assay, resulted in a time dependent decrease in [3H]-thymidine incorporation into DNA and MTT formazan formation, respectively. The cell cycle regulation measured by flow cytometry with propidium iodide staining revealed that the cell multiplication cycle was slowed down due to accumulation of cell in S-phase of the cell cycle, leading to inhibition in G0/G1 phase of the cell cycle. The 5-LO protein expression (measured by Western immunoblot analysis) and LTB4 biosynthesis (measured by enzyme immunoassay) were found to be increased by 2 to 2.4 fold and several fold respectively on days 3 and 4 of MuIFN-alpha/beta treatment. The GFAP protein expression was also found to be increased at least by 3 fold on day 4 of the MuIFN-alpha/beta treatment. These results suggest that inhibition in growth and thereby slowing of the cell multiplication cycle of glioma cells has resulted in upregulation of GFAP expression and 5-LO pathway.

Inhibition of human glioma cell proliferation and glutathione S-transferase by ascorbyl esters and interferon.

The in vitro effect of ascorbyl esters (ascorbyl-stearate [As-S] and -palmitate [As-P]) and interferon (recombinant human interferon-a2b [rHuIFN-a2b]) on human glioma (U-373) cell proliferation, viability and glutathione-S-transferase (GST) activity was studied. The effect of As-S, As-P and rHuIFN-a2b on cell proliferation and viability was evaluated by [3H] Thymidine incorporation and colorimetric MTT assays, respectively. Incubation of glioma cells with As-S, As-P or rHuIFN-a2b for 24 h resulted in a dose dependent inhibition of cell proliferation (IC50 = 68.0 microM As-S, 86.0 microM As-P and 47.3 Units/ml rHuIFN-a2b), and moderate decrease of cell viability. It was found that As-S was a more efficient inhibitor of cell proliferation, viability and GST activity than As-P. GST from U-373 cells was purified. The activity of purified GST towards 1-chloro-2,4-dinitrobenzene (CDNB) was inhibited in a dose dependent manner by ascorbyl esters (I-50 = 27.5 microM As-S and 56.0 microM As-P) but not by rHuIFN-a2b. GST activity of cytosol isolated from U-373 cells which were previously treated with As-S (150 microM) or rHuIFN-a2b (150 units/ml) for 0, 2, 5, 10, 20 and 30 min was sharply decreased during 5 to 10 min of treatment and increased at longer durations of treatment.

Interferon entry through the blood-brain barrier in glioma and its effect on lipoxygenase activity.

This study evaluates cerebral entry of mouse interferon alpha/beta (MuIFN alpha/beta) or mouse interferon gamma (MuIFN-gamma) following continuous (3 day), subcutaneous infusion of normal or glioma bearing mice. The intracerebral C57BL/6 mouse glioma-26 (G-26) model was used at days 10-14 post tumor implant, the advanced stage of glioma progression as defined by histology and the median survival time (27 +/- 3.8 days). The infusion of horseradish peroxidase (HRP) in vivo at day 10 or 11 post glioma implant showed strong staining in the tumor bed indicating compromised blood-brain barrier (BBB). In addition, histochemistry with Bandeiraea simplicifolia isolectin B4 demonstrated the accumulation and/or activation of macrophage/microglia. The 3 day infusion of mice (day 11-14 post tumor implant) via subcutaneous (sc) osmotic micro-pumps with MuIFN alpha/beta (8x10(5) - 1.7x10(6) international units [IU]/ml) or with recombinant mouse interferon gamma (rMuIFN-gamma) (1x10(6) IU/ml) resulted in a low but detectable (1-5 IU/ml) cerebral level of IFN. The IFN levels in the blood (20-40 IU/ml) and brain, measured by assay of inhibition of viral cytopathic effect (CPE) or ELISA assay for MuIFN-gamma, showed no difference between normal and glioma bearing mice. The lipoxygenase (LO) activity (dioxygenase) of glioma tissue and contralateral control was evaluated in non-treated and MuIFN alpha/beta continuously (3 day) treated mice. The LO activity in glioma tissue was significantly higher (p < 0.05) than the contralateral control in non-treated mice. However, following sc MuIFN alpha/beta infusion the LO activity of glioma decreased to control level.

Brain CT and MRI findings after carbon monoxide toxicity.

The neuropathologic sequelae of carbon monoxide (CO) toxicity have been well described in postmortem examinations. Globus pallidus damage as well as diffuse white matter lesions and encephalopathic changes occur. Brain CT has provided imaging correlates to the premortem changes. MRI is more sensitive and provides more specificity. Cerebral edema changes may occur early with subsequent demonstration of globus pallidus lesions and white matter changes. Globus pallidus lesions in many cases do not correlate directly to clinical status and outcome; however, the presence of diffuse white matter disease is a more reliable index of both. These changes are seen in patients in both accidental exposures to CO and in suicide attempts.

Perfusion and molecular modification of idoxuridine to alter its cerebrospinal fluid metabolism.

Two methods to deter the rapid intrathecal degradation of idoxuridine were investigated: (a) rapid drug perfusion through the ventricular system, and (b) modification of the molecule to its uronic acid derivative, 2'-deoxy-5-iodo-5'-uridinecarboxylic acid, to make it less susceptible to enzymatic digestion. Perfusion of idoxuridine through the ventricular system (ventriculocisternal) of dogs at 0.97 ml/min saturated the metabolic pathway so that the outflow solution yielded a single spot (Rf 0.76) on TLC indicative of the intact molecule. The 125I-labeled uronic acid was synthesized from the 125I-labeled parent compound, and the labeled compounds were compared after their individual intracisternal injection in dogs. Since there was no difference in the disappearance rates, the stability of the uronic acid was, in fact, no greater than that of the parent compound in vivo. Ventricular perfusion of idoxuridine, however, seems a suitable means for increasing the amount of active drug delivered to central nervous system tumors and viral infections.

Pathology of lacquer thinner induced neuropathy.

Seven men aged 17 to 22 years developed severe distal symmetrical predominately motor polyneuropathy after repeated inhalation of a commercially available brand of lacquer thinner. Motor nerve conduction velocities were markedly slowed. Fascicular biopsy specimens of sural nerve showed a striking loss of myelinated nerve fibers. Prominent neurofilamentous masses resulted insegmental paranodal distention of axons with secondary thinning and retraction of myelin from the node of Ranvier. Autopsy material from one case revealed central chromatolysis of anterior horn cells in the lumbosacral enlargement and axonal swellings in the fasciculus gracilis. One or more volatile hydrocarbons contained in the lacquer thinner involved may be neurotoxic if inhaled to excess any many cause a neuropathy with characteristic pathological features.

Entry rate kinetics of D-mannitol and carboxyl-inulin for transfer across the blood-cerebrospinal fluid barrier.

This study evaluates the entry rate kinetics of hydrophilic compounds [3H]-D-mannitol and [14C]-carboxyl-inulin across the blood-cerebrospinal fluid (CSF) barrier in a rabbit experimental model. To maintain steady state levels of these tracers in circulation, 100 microCi of [3H]-D-mannitol and 150 microCi of [14C]-carboxyl-inulin were administered as a bolus and by slow infusion for four hours via a femoral venous catheter. Entry rate kinetics of [3H]-D-mannitol and [14C]-carboxyl-inulin from plasma into cisterna magna CSF were computed using a mathematical equation described by Davson. [3H]-D-mannitol and [14C]-carboxyl-inulin maintained steady state levels throughout the experiment. Entry rates for mannitol and carboxyl-inulin were represented by a straight line, from the slope of which K(out) (or K(in)) were computed: K(in) values for mannitol and carboxyl-inulin were 0.06820 hr(-1) and 0.00023 hr(-1), respectively. Differences in the entry rate of mannitol and carboxyl-inulin may be explained by the molecular size and effective radius of these tracers.

Selective permeability of [3H]-D-mannitol and [14C]-carboxyl-inulin across the blood-brain barrier and blood-spinal cord barrier in the rabbit.

Selective permeability across the blood-brain and blood-spinal cord barriers was studied in a rabbit experimental model. To maintain steady state levels of the tracers in the circulation, 50muCi each of [3H]-D-mannitol and [14C]-carboxyl-inulin were administered as a bolus and by slow intravenous infusion for 60, 90 and 120 minutes; 90 min proved to be the optimal equilibration time for uptake of radioactive tracers into central nervous system (CNS) regions. Kinetic transfer of [3H]-D-mannitol and [14C]-carboxyl-inulin from blood into CNS was computed as apparent transfer constant (KD). The KD for [3H]-D-mannitol in regions of brain (cerebrum, cerebellum, mid-brain, pons and brain stem), and spinal cord (cervical, thoracic and lumbar) were 0.033 to 0.054 microliter/min/g-1 and 0.053 to 0.065 microliter/min/g-1, respectively. The KD for [14C]-carboxyl-inulin into brain and spinal cord regions were 0.016 to 0.033 and 0.037 to 0.054 microliter/min/g-1, respectively. Of the regions of spinal cord and brain examined, lumbar cord appears to be the most permeable. The KD values pooled for samples of the spinal cord show a significant increase in uptake of [3H]-D-mannitol (p = 0.0043) and [14C]-carboxyl-inulin (p = 0.0001) compared to samples of brain. The blood-spinal cord barrier was more permeable to these substances than the blood-brain barrier.

Epinephrine increases the selective permeability of epidurally administered [3H]-D-mannitol and [14C]-carboxyl-inulin across the blood-spinal cord barrier.

A rabbit experimental model was used to ascertain how epinephrine influences uptake of epidurally-administered [3H]-D-mannitol and [14C]-carboxyl-inulin into regions of brain and spinal cord. [3H]-D-mannitol and [14C]-carboxyl-inulin, 20 microCi each, were distributed in normal saline (control) and normal saline with 0.03 microM (1:200,000 diluted) epinephrine. These tracers were administered as bolus and slow epidural infusion for 90 min. Epinephrine decreased the uptake of [3H]-D-mannitol and [14C]-carboxyl-inulin into the serum collected at 15, 30, 45, 60, 75 and 90 min intervals during the administration of tracers. Epinephrine did not alter the uptake of [3H]-D-mannitol and [14C]-carboxyl-inulin into the regions of the brain (cerebrum, cerebellum, brain stem including midbrain and pons) and upper regions of the spinal cord (mid-cervical and mid-thoracic) compared to controls. The uptake of [3H]-D-mannitol and [14C]-carboxyl-inulin into the mid-lumbar region is significantly increased compared to the control. The differential uptake of [3H]-D-mannitol and [14C]-carboxyl-inulin into 1 cm thick sections of lumbo-sacral cord was significantly increased by epinephrine at the site of the epidural catheter placement, while sections of the cord distal on either end (thoracic and sacral ends) did not show significant differences compared to the control. The efficacy of epinephrine in increasing spinal cord uptake of hydrophilic [3H]-D-mannitol and [14C]-carboxyl-inulin may be attributed to its vasoconstrictive properties and its ability to increase vascular endothelial cell permeability across the blood-spinal cord barrier.