Joseph P. Hanig

Neuroprotective effect of the chemical chaperone, trehalose in a chronic MPTP-induced Parkinson's disease mouse model

Parkinsons disease (PD) is a progressive motor disease of unknown etiology in the majority of cases. The clinical features of PD emerge due to selective degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), which project to the caudate putamen (CPu) where they release DA. In the current in vivo mouse model study, we tested trehalose for its ability to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage to DA neurons. Trehalose is a naturally occurring disaccharide present in plants and animals and appears capable of protecting cells against various environmental stresses. The effect of trehalose is likely due to its action as a pharmacological chaperone which promotes protein stability. In the present study, there were four treatment groups: saline only (control); probenecid only; MPTP+probenecid; and trehalose+MPTP+probenecid. MPTP-induced losses in tyrosine hydroxylase and DA transporter immunoreactivity in the ventral midbrain SNc and CPu were significantly reduced by trehalose. Decreases in CPu dopamine levels produced by MPTP were also blocked by trehalose. Microglial activation and astrocytic hypertrophy induced by MPTP were greatly reduced by trehalose, indicating protection against neuroinflammation. These effects are commensurate with the observed trehalose sparing of motor deficits produced by MPTP in this mouse model. Two tight junctional proteins, ZO-1 and occludin, are downregulated following MPTP treatment and trehalose blocks this effect. Likewise, the glucose transporter-1 that is expressed in brain endothelial cells is also protected by trehalose from MPTP-induced down-regulation. This study is the first to demonstrate using fluoro-turoquoise FT gel perfusion techniques, the protection afforded by trehalose from MPTP-induced damage to microvessels and endothelial and suggests that trehalose therapy may have the potential to slow or ameliorate PD pathology.

Ethanol enhancement of blood-brain barrier permeability to catecholamines in chicks

Abstract The effect of ethanol (EtOH) on permeability of the blood-brain barrier (BBB) to parenteral catecholamines was investigated in neonate chicks. Animals simulataneously administered EtOH, 2 g/kg, and norepinephrine (NE), 5 mg/kg, or epinephrine (E), 5 mg/kg, entered a roosting state which was more pronounced than that observed after either amine alone. Roosting chicks were killed 2 min after NE + EtOH and 10 min after E + EtOH for whole brain amine analysis. NE + EtOH treatment resulted in a 220% increase in whole brain NE over controls receiving this amine alone, whereas E + EtOH produced a 29% increase in brain E. EtOH alone did not alter endogenous levels of either amine in brain. Results indicate that EtOH, a solvent whose solubility characteristics allow it to penetrate easily both aqueous and lipoid membrane components, facilitates entry of E and NE into the brain of the neonate chick across an imperfect BBB present at hatching.

Increase of blood-brain barrier permeability to catecholamines by dimethyl sulphoxide in the neonate chick

Parenteral adrenaline or noradrenaline, because they cross a permeable blood-brain barrier in young chicks, produce sustained roosting or sleeping behaviour (Key & Marley, 1962; Spooner, Mandell & others, 1968; Hanig & Seifter, 1968). This phenomenon disappears within 6-8 weeks as the various components of the central nervous system mature (Waelsch, 1955 ; Lajtha, 1957). Several years ago, Brink & Stein (1967) showed that dimethyl sulphoxide (DMSO) facilitated the entry of [14C]pemoline into the brain of rats. Recently, De La Torre (1970) has shown that DMSO increases the relative penetration of L-dopa and 5-HTP across the blood-brain barrier of the rat. DMSO is an industrial solvent with skin-penetrating effects, and the ability to carry into the body toxic substances that are normally excluded (Stoughton & Fritsch 1964; Kligman, 1965; Narula, 1967). Since DMSO has both aqueous and lipid solubility characteristics that allow it to penetrate the central nervous system easily, it was chosen as a prototype compound for study of blood-brain barrier biogenic amine interaction in the neonate chick. We observed that DMSO intensified roosting induced by adrenaline and noradrenaline in the neonate, and therefore sought to determine whether this was associated with increased penetration of catecholamines into chick brain (Hanig, Morrison, & Krop, 1970). One-day-old chicks housed in a temperature-controlled brooder were given free access to food and water and used within one week. Adrenaline or noradrenaline was dissolved in 0.9% NaCl or 50% DMSO--O.9% NaCl and administered (5 mg/kg, i.v.) into the jugular vein. This corresponded to a dose of 2.75 g/kg of DMSO which is well below the single dose toxicity for various species described by Smith, Hadidian & Mason (1967). Chicks were decapitated 10 min after adrenaline administration, but 2 min after noradrenaline because of its rapid turnover in the brain. Both amines in whole brain were estimated by an automated fluorimetric procedure (Hanig, Morrison & Krop, 1969). The grossly observable behaviour of DMSO alone in chicks was minimal except for a transitory arousal that lasted for several seconds after injection, whereas those receiving adrenaline or noradrenaline with DMSO exhibited a roosting response that was more intensified than that observed with the same dose of either amine alone. Preliminary chemical studies showed no difference in endogenous amine concentra tions between animals treated with saline or those treated with DMSO alone (Hanig & others, 1970). Administration of adrenaline + DMSO gave a highly significant increase of 34.8% in concentrations of this amine in brain over controls receiving the same dose of adrenaline alone. Similarly, noradrenaline + DMSO treatment gave

Toxicity of botulinum toxin: A stoichiometric model for the locus of its extraordinary potency and persistence at the neuromuscular junction

Abstract The number of botulinum toxin molecules calculated to reach either a diaphragmatic endplate after a minimum lethal dose or a gastrocnemius endplate after a minimal paralyzing dose is in close agreement with both the number of vesicles released per impulse and the finite number of active sites on the endplate for efflux of acetylcholine-containing vesicles. The close agreement in size between botulinum toxin molecules, synaptic vesicles, and the width of neurotubules supports a model in which botulinum toxin physically blocks efflux of acetylcholine. The stoichiometric attachment of botulinum toxin may occur at the critical moment that vesicles fuse with the presynaptic membrane, thereby exposing a receptor. While attachment may be to a ganglioside or neuraminidase-sensitive site, the chemical nature of the receptor is neither defined nor critical for the validity of the model.

Long-Term Consequences of Drugs on the Paediatric Cardiovascular System

Many pharmacological and toxicological actions of drugs in children cannot be fully predicted from adult clinical experience or from standard non-clinical toxicology studies. Numerous drugs have direct or indirect pharmacological effects on the heart and are prescribed for children of all ages. Toxicity or secondary effects may be immediate or delayed for years after drug exposure has ceased. Originally, the aim of this review was to compile information on the effect of specific drugs on the post-natal development of the cardiovascular system and to examine long-term follow-up of the use of cardio-active drugs in children. The limited database of published information caused the original question to evolve into an examination of the medical literature for three areas of information: (i) whether vulnerable developmental windows have been identified that reflect the substantial functional development that the cardiovascular system undergoes after birth; (ii) what is known about pharmacological perturbation of development; and (iii) what the likelihood is of drug exposure during childhood. We examined different scenarios for exposure including random, isolated exposure, conditions historically associated with adults, primary or secondary cardiac disease, psychiatric and neurological conditions, asthma, cancer and HIV. Except for random, isolated drug exposures, each category of possible exposure contained numerous drugs known to have either primary or secondary effects on the cardiovascular system or to influence factors associated with atherosclerosis. It is likely that a significant number of children will be prescribed drugs having either direct or indirect effects upon the immature cardiovascular system. A confounding factor is the simultaneous use of over-the-counter medications and herbal or nutraceutical preparations that a patient, parent or guardian does not mention to a prescribing physician. Metabolism is also important in assessing drug effects in children. Differences in body water: body fat ratio, age-related gastrointestinal absorption, distribution, excretion, renal function and drug metabolizing capabilities make it possible for children to have a different metabolite profile for a drug compared with adults. There is little examination of drug effects on the interdependent processes of cardiac maturation and less examination of metabolite effects. It is difficult to identify delayed toxicities in children as these adverse events may take years to manifest with many patients lost to follow-up. Clearly this is an area of study where intermediate endpoints and surrogate markers would be of great benefit. Pharmacogenomics may be useful in providing markers of increased risk or susceptibility. A perspective must be kept in balancing the possibility of a problem with the very real benefits that many children experience from the use of these pharmaceuticals.

Increased neurobehavioral toxicity of styrene in protein-malnourished rats

Influence of protein deficiency on the neurobehavioral toxicity of styrene during gestation and early infancy was studied in rats. Eye opening and fur growth were delayed in rat pups born to dams receiving a low protein diet. These pups also showed a delay in the development of surface and air righting reflexes and cliff avoidance response and a marginal increase in the levels of dopamine and serotonin receptors in comparison to those born to dams receiving a normal protein diet. Alterations in these parameters were more marked in pups born to dams exposed to styrene and receiving a low protein diet. In addition, these pups also showed a significant decrease in the activity of monoamine oxidase, Na+, K(+)-ATPase and succinic dehydrogenase as well as significant increases in motor activity and receptor sensitivity when compared to rat pups born to dams receiving a low protein diet. No significant alterations in behavioral and biochemical parameters were observed in the pups born to dams exposed to styrene and receiving a normal protein diet at this dose level. These results suggest that protein deficiency during early life renders the animals more susceptible to styrene.

Convulsions in weanling rabbits after a single topical application of 1% lindane

Abstract There have been sporadic reports of adverse effects of lindane (γ-hexachlorocyclohexane) in children treated for scabies. These effects usually consisted of tremors, convulsions, anorexia, and possible stunting of growth. Weanling rabbits (6 weeks old; 1.0 kg) given a single topical application of 1% lindane at a dose reportedly used in infants (60 mg/kg) exhibited severe anorexia and convulsions; death occurred in some cases. The effects were more pronounced in weanlings in which the skin was inflamed or not completely intact. In contrast, young adult rabbits (2–3 kg) given the same dose showed only some anorexia and possibly mild excitement. Concentrations of lindane in whole blood of weanlings at the time of convulsion (approximately 24 hr after dosing) ranged from 0.7 to 2.5 μg/ml. These findings suggest a rather pronounced sensitivity of weanlings to lindane in comparison to adults.

Observations on hexachlorophene-induced paralysis in the cat and its antagonism by hypertonic urea.

Summary Cats given HCP (20 mg/kg) orally each day developed hindlimb paralysis and greatly elevated CSFP (174 mm saline; 19 mm in controls) in 3 to 5 days. “Status spongiosis” was seen in white matter only in sections of brain and cord. There was no dilation of cerebral ventricles, or damage to their ependymal linings or to the arachnoid villi. The neurological picture excluded any but a terminal effect upon cranial nerve function. There appeared to be no damage to neurons, and recovery of survivors was complete within 6 weeks after cessation of HCP administration. Elevated CSFP in paralyzed anesthetized cats was quickly lowered by an average of 256 mm by slow iv administration of 30% urea (2 g/kg in 10% invert sugar). Unanesthetized cats similarly paralyzed were able to stand and walk for up to 4 hr after this treatment. Neither acetazolamide nor prednisolone alone had any effect, nor did coadministration with urea enhance the effect of urea. The HCP lesion does not appear to be inflammatory in origin, nor does it seem to involve ventricular obstruction or overproduction of cerebrospinal fluid. The reappearance of paralysis about 4 hr after osmotic diuresis, which corresponds with the elimination of urea, suggests that prolonged iv infusion with urea or a similar osmotically active substance may have significant clinical value in the management of HCP poisoning. We wish to thank Dr. Renate D. Kimbrough of the Center for Disease Control for her generous assistance and expert opinion on a portion of the preliminary neuropathology studies; Dr. Joseph Fenstermacher and his staff of the National Institutes of Health for their excellent advice on the performance of cerebrospinal fluid measurements; Dr. Andrew Ulsamer of the Consumer Product Safety Commission for expert guidance and assistance with the hexachlorophene analytical procedure; and Dr. Rogelio A. Zaldivar of the Food and Drug Administration for the excellent veterinary care of our animal colony.

Protection with butylated hydroxytoluene and other compounds against intoxication and mortality caused by hexachlorophene

The antioxidants butylated hydroxytoluene (BHT) and ethoxyquin protected rats against intoxication and mortality normally produced by hexachlorophene (HCP, 100 mg/kg). BHT also prevented the elevation of cerebrospinal fluid pressure, a central nervous system effect of HCP poisoning. In addition, both phenobarbital and SKF-525A protected against HCP poisoning, with the barbiturate also offering significant protection against triethyltin. L-Ascorbic acid, vitamin E, N,N-diphenyl-p-phenylenediamine and reduced and oxidized glutathione over a range of doses were ineffective in preventing HCP lethality. The protective effect of phenobarbital against HCP and triethyltin intoxication further supports existing evidence of a common or similar mechanism of toxic action for these two structurally dissimilar compounds.

Alteration of Sensitivity of Adrenergic Vascular Responses after Prolonged Exposure to Agonists via Osmotic Minipump

Abstract A study was performed to determine whether a constant 1-week exposure to either alpha or beta agonists in vivo would allow alteration or manipulation of the responses of rat aortic alpha- and beta-adrenergic receptors. Osmotic minipumps delivering either phenylephrine, isoproterenol, or propranolol for 7 days at a dose of 3.2, 4.2, or 5.2 mg/kg/day, respectively, were implanted in male Holtzman rats under halothane anesthesia. Seven days later, rats were killed and aortic ring preparations were used to measure alpha- and beta-adrenergic responses. In phenylephrine-pretreated rats, alpha-adrenergic responses, as measured by contractions induced by phenylephrine, were markedly reduced (P < 0.05) across a dose range of 10-9 to 10-6 M. In contrast, in these same phenylephrine-pretreated preparations, the beta-adrenergic responses involving isoproterenol-induced relaxation were significantly increased (P < 0.05) across a dose range of 10-7 to 10-5 M. Isoproterenol pretreatment for 7 days resulted in a statistically significant reduction of beta-adrenergic aortic relaxation, whereas the alpha-adrenergic responses to phenylephrine remained unchanged compared with controls. Propranolol pretreatment had no effect on either alpha- or beta-adrenergic responses. These findings indicate that the alpha agonist-induced response after in vivo pretreatment induces reciprocal changes in the functionally related beta-adrenergic apparatus, and also suggest linkage between these two receptors. In contrast, the beta response appears to desensitize or downregulate in response to beta agonist exposure in a manner that seems to be independent of or to operate in the absence of an alteration of the alpha response.