Modelling Parkinson-like neurodegeneration via osmotic minipump delivery of MPTP and probenecid

Mouse models of MPTP intoxication have been used extensively to explore the molecular mechanisms of Parkinson's disease. However, these models present some limitations since; (i) Dopaminergic (DA) cell death occurs rapidly in contrast to the presumably slow evolution of the disease process. (ii) Some of the key histological features of the disease such as Lewy body like inclusions and long-term inflammatory changes are lacking. Fornai et al. [Proc. Natl Acad. Sci. USA 102 (2005), 3413] suggested that continuous delivery of MPTP with Alzet osmotic minipumps may possibly circumvent these problems. Our results show, however, that MPTP infusion via Alzet osmotic minipumps (40 mg/kg/day) produces only a transient depletion in striatal dopamine (DA) without causing dopaminergic cell loss in the substantia nigra. Neuronal cell loss occurred, however, if MPTP was infused concomitantly with probenecid, an uricosuric agent which potentiates the effects of the toxin injected via the i.p. route. Even under these conditions, dopaminergic cell loss was moderate (-25%) and other neurodegenerative changes characteristic of Parkinson's disease remained undetectable.     

In vitro oxidation of MPTP by primate neural tissue: A potential model of MPTP neurotoxicity

The compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a parkinsonian syndrome in humans and primates. We have previously found that metabolism of MPTP to a quaternary species is necessary for the expression of its neurotoxic effects. We now report that the metabolism of MPTP occurs in primate brain tissue $\text{in vitro}$, and present a model of MPTP neurotoxicity which incorporates our findings to date.Since the toxicity of MPTP is metabolism dependent, we propose that the $\text{in vitro}$ metabolism of MPTP by brain tissue should provide a useful model for studying selected aspects of MPTP neurotoxicity.     

Protocol for the MPTP mouse model of Parkinson's disease

This protocol describes our method of producing a reliable mouse model of Parkinson's disease (PD) using the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We discuss the particulars of the model, provide key references and outline what investigators need to know to develop the MPTP mouse model of PD safely and successfully. Completion of this protocol depends on the regimen of MPTP used and on the actual planned studies, which often range from 7 to 30 d. This protocol calls for implementation of safety measures and for the acquisition of several pieces of equipment, which are a one-time investment worth making if one elects to use this model on a regular basis.     

A parkinsonian syndrome induced in the goldfish by the neurotoxin MPTP.

Parkinson's disease has been modeled in humans, lower primates, and to a lesser extent in some other vertebrates by administration of the potent neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine). The MPTP model has thus drawn considerable attention as a system to search for anti-Parkinson's disease drugs, although the cost and scarcity of primates has limited extensive applications. We now report that a parkinsonian syndrome can be elicited in the common goldfish (Carassius auratus) by a single dose of MPTP. The syndrome is characterized by profound bradykinesia (slow movement), the full extent of which is reached 3 days after MPTP administration. The reduction in movement is paralleled by loss of dopamine and norepinephrine from the forebrain and midbrain and in other brain regions as well. The toxic oxidative product of MPTP, MPP+, is also accumulated predominantly in forebrain and midbrain, and pretreatment with the monoamine oxidase blocker tranylcypromine substantially reduces accumulation of the toxic metabolite. A barely perceptible coarseness in balance adjustment also occurs in treated animals. The MPTP-treated goldfish recover normal movement and normal brain monoamine levels within 10-13 days after administration of the drug. We interpret these and other data to indicate that MPTP can induce a Parkinson's disease-like syndrome in the goldfish that is similar in many aspects to the syndrome induced by MPTP in humans and other primates. This remarkable parallel may permit the goldfish to supplement expensive and scarce primates for the purpose of searching and screening neuroprotective drugs with specific relevance to Parkinson's disease.     

Neuroprotective effects of ginkgetin against neuroinjury in Parkinson's disease model induced by MPTP via chelating iron

Abstract

Disruption of neuronal iron homeostasis and oxidative stress are closely related to the pathogenesis of Parkinson's disease (PD). Ginkgetin, a natural biflavonoid isolated from leaves of Ginkgo biloba L, has many known effects, including anti-inflammatory, anti-influenza virus, and anti-fungal activities, but its underlying mechanism of the neuroprotective effects in PD remains unclear. The present study utilized PD models induced by 1-methyl-4-phenylpyridinium (MPP⁺) and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to explore the neuroprotective ability of ginkgetin in vivo and in vitro. Our results showed that ginkgetin could provide significant protection from MPP⁺-induced cell damage in vitro by decreasing the levels of intracellular reactive oxygen species and maintaining mitochondrial membrane potential. Meanwhile, ginkgetin dramatically inhibited cell apoptosis induced by MPP+ through the caspase-3 and Bcl2/Bax pathway. Moreover, ginkgetin significantly improved sensorimotor coordination in a mouse PD model induced by MPTP by dramatically inhibiting the decrease of tyrosine hydroxylase expression in the substantia nigra and superoxide dismutase activity in the striatum. Interestingly, ginkgetin could strongly chelate ferrous ion and thereby inhibit the increase of the intracellular labile iron pool through downregulating L-ferritin and upregulating transferrin receptor 1. These results indicate that the neuroprotective mechanism of ginkgetin against neurological injury induced by MPTP occurs via regulating iron homeostasis. Therefore, ginkgetin may provide neuroprotective therapy for PD and iron metabolism disorder related diseases.     

Parkinson-like disease by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity inMacaca Fascicularis: Synaptosomal metabolism and action of dihydroergocriptine

The maximal rates (V_max) of some enzyme activities related to synaptosomal energy metabolism were studied in different types of synaptosomes from cerebellar cortex ofMacaca Fascicularis (Cynomolgus monkey). Different synaptosomal populations, namely large and small synaptosomes, were isolated from the anterior lobule of the cerebellar cortex of monkeys treated p.o. with dihydroergocriptine at the dose of 12 mg/kg/day before and during the induction of a Parkinson's-like syndrome by MPTP administration (i.v., 0.3 mg/kg/day for 5 days). The enzymes were chosen according to their regulatory role and as markers of the following metabolic pathways: (a) glycolysis ((hexokinase, phosphofructokinase, lactate dehydrogenase), (b) Krebs' (TCA) cycle (citrate synthase, malate dehydrogenase), (c) amino acid, glutamate metabolism (glutamate dehydrogenase, glutamate-pyruvate- and glutamate-oxaloacetate-transaminases), (d) acetylcholine catabolism (acetylcholinesterase) and (e) ATPases, i.e. Na⁺–K⁺-ATPase, Mg²⁺-ATP synthetase, Mg²⁺-ATPase, Ca²⁺–Mg²⁺-ATPase and Ca²⁺-ATPase Low and High affinity for Ca²⁺. The MPTP administration modified the activities of citrate synthase, malate dehydrogenase, Na⁺–K⁺-ATPase, acetylcholinesterase and glutamate-oxaloacetate transaminase only on selected types of synaptosomes.Pharmacological treatment by dihydroergocriptine was able to recovery at the steady-state levels the activities of these enzymes, thus demonstrating a partial protective effect on these biochemical parameters.     

A sheep cannulation model for evaluation of nasal vaccine delivery

We have developed and validated a novel model to investigate the efficacy of nasal vaccine delivery. Based on lymphatic cannulation of the tracheal lymph trunk of sheep, the model allows collection of lymph draining from the Nasal Associated Lymphoid Tissue. The model is suitable for determining both the amount of material that is absorbed into the lymphatic system, following intra-nasal delivery and the immune response that occurs following vaccination into the nasal area. The cell populations that track in this duct were phenotyped and found to be similar to those previously reported to be present in efferent lymph draining from peripheral lymph nodes. Following intra-nasal spray, we demonstrated that the amount of material recovered in draining lymph is only a very small fraction of the total delivered. Nevertheless, intra-nasal spraying of a vaccine could activate local immune cells. The method described will be invaluable for optimising intra-nasal delivery systems by allowing a separate optimisation of vaccine uptake and immune responses induction.     

A simple model for calcium induced exocytosis

We have developed a simple model showing how the presence or absence of Ca²⁺ can determine whether an uncurved or curved membrane surface is favored energetically. The model shows why fusion of vesicles with the presynaptic membrane is favored in the presence of calcium and why the budding off of vesicles is favored in the absence of calcium inside of the presynaptic membrane. The model accurately predicts the radius of a synaptic vesicle using known properties of lipids and suggests consequences of temperature change, varied stimulation rate and addition of calcium by artificial means on rates of transmitter release.     

A catalyst function for MPTP in superoxide formation

We demonstrate that 1-methyl-4-phenyl-1,2-dihydropyridine (MPDP) can be generated, in an alternate pathway, from the catalyst action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon the iron redox equilibrium reaction. Superoxide and ferric iron are instantaneously produced after addition of MPTP to a solution of ferrous iron. This reaction is oxygen and pH dependent. Superoxide, through a iron dependent Haber-Weiss reaction with peroxide, can generate the cytotoxic hydroxyl radical. A small portion of the superoxide reacts with MPTP to produce the reactive species X. which, in the presence of Fe+3 can also generate MPDP.     

Neurodegenerative alterations induced by MPTP neurotoxin in senescence accelerated mice essay

Behavioral modifications and alterations in biochemical pathways induced by neurotoxin MPTP in Senescence Accelerated Mice (SAM) brains are discussed. MPTP injections lead to specific injuries of dophaminergic neurons and to reinforcement of oxidative stress conditions. The ability of neuropeptide carnosine to protect animals from oxidative injuries induced by MPTP injections is also described.     

A sheep model for fracture treatment in osteoporosis: benefits of the model versus animal welfare

Animal models are necessary to evaluate new options for the treatment of fractures in osteoporotic bone. They permit both the biological response of a living system and the influence of the pathological processes to be taken into account. A sheep model for osteoporosis was established by combining oestrogen deficiency, calcium and vitamin D-deficient diet with steroid medication. Bone mineral density (BMD) was reduced by >30% after 12 weeks of combined treatment. Osteoporosis similar to the human situation with corresponding changes in the micro-architecture and mechanical properties of bone was observed. This publication focuses on the impressive results obtained with the model and contrasts them with considerations of animal welfare. Considerable side-effects associated with steroid medication became manifest. Animals in the treatment groups showed signs of infection of various degrees due to the immunosuppressive effect of the medication. The infections were mostly caused by Corynebacterium pseudotuberculosis. Antibody testing revealed a 100% prevalence of infection in this breed of sheep. A modification of the steroid treatment, i.e. less-frequent injections, reduced the incidence of side-effects. This sheep model shows a significant and reproducible reduction in cancellous BMD of >30%, including relevant changes in biomechanical properties and increased fracture risk. However, the severity of the side-effects cannot be overlooked. The model must be improved if it is to be used in the future. Options to reduce the side-effects are discussed.     

A dynamic model for induced reactivation of latent virus

We develop a deterministic mathematical model to describe reactivation of latent virus by chemical inducers. This model is applied to the reactivation of latent KSHV in BCBL-1 cell cultures with butyrate as the inducing agent. Parameters for the model are first estimated from known properties of the exponentially growing, uninduced cell cultures. Additional parameters that are necessary to describe induction are determined from fits to experimental data from the literature. Our initial model provides good agreement with two independent sets of experimental data, but also points to the need for a new class of experiments which are required for further understanding of the underlying mechanisms.     

Cyclosporine A suppresses keratinocyte cell death through MPTP inhibition in a model for skin cancer in organ transplant recipients

Transplant recipients have an elevated risk of skin cancer, with a 65- to 250-fold increase in squamous cell carcinoma. Usage of the immunosuppressant cyclosporine A (CsA) is associated with the development of skin cancer. We hypothesized that the increased incidence of skin cancer was due to the action of CsA within keratinocyte mitochondria where it can inhibit mitochondrial permeability transition pore (MPTP) opening. Normally, MPTP opening is induced by oxidative stress such as that caused by UV light and leads to cell death, thereby eliminating a cell that has been exposed to genotoxic insult. However, in the presence of CsA, damaged cells may survive and consequently form tumors. To test this hypothesis, we treated keratinocytes with levels of CsA used therapeutically in transplant patients and assessed their viability following UVA-irradiation. CsA prevented cell death by inhibiting MPTP opening, even though the levels of oxidative stress were increased markedly. Nim811, a non-immunosuppressive drug that can block the MPTP had a similar effect while the immunosuppressive drug tacrolimus that does not interact with the mitochondria had no effect. These findings suggest that CsA may promote skin cancer in transplant patients by allowing keratinocyte survival under conditions of increased genotoxic stress.     

A model for continuous production of thermally induced recombinant proteins

Summary General conditions for continuous expression of heterologous genes fromP _L promoter in two fermenters connected in series have been established. The induction time of the bacterial cells is calculated as a function of the retention time in the inducing reactor. Using this model, it is possible to adapt fermentation parameters to the particular behaviour of any specific recombinant clone.Nomenclature F(t) flow at timet [ml/min] - M _T (t) culture induced, at timeT of fermentation, during a period up tot [ml] - N _T (t) culture induced, at timeT of fermentation, during a period fromt tot+dt [ml] - p(t) product yield in a discontinuous culture [units/ml] - P(t) product yield at the outlet of the fermenter [units/ml] - v(t) volume of culture entered into the inducing reactor up to timet [ml] - V volume of the inducing reactor [ml] Greek letters retention time in the inducing reactor [min] - (t) average induction time at timet [min]     

Posterolateral spinal fusion with ostegenesis induced BMSC seeded TCP/HA in a sheep model

Autogenous bone graft is the gold standard for fusion procedure. However, pain at donor site and inconsistent outcome have left a surgeon to venture into some other technique for spinal fusion. The objective of this study was to determine whether osteogenesis induced bone marrow stem cells with the combination of ceramics granules (HA or TCP/HA), and fibrin could serve as an alternative to generate spinal fusion. The sheep's bone marrow mesenchymal stem cells (BMSCs) were aspirated form iliac crest and cultured for several passages until confluence. BMSCs were trypsinized and seeded on hydroxyapatite scaffold (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) for further osteogenic differentiation in the osteogenic medium one week before implantation. Six adult sheep underwent three-level, bilateral, posterolateral intertransverse process fusions at L1–L6. Three fusion sites in each animal were assigned to three treatments: (a) HA constructs group/L1–L2, (b) TCP/HA constructs group/L2–L3, and (c) autogenous bone graft group/L5–L6. The spinal fusion segments were evaluated using radiography, manual palpation, histological analysis and scanning electron microscopy (SEM) 12 weeks post implantation. The TCP/HA constructs achieved superior lumbar intertransverse fusion compared to HA construct but autogenous bone graft still produced the best fusion among all.     

PACAP27 prevents Parkinson-like neuronal loss and motor deficits but not microglia activation induced by prostaglandin J2

Neuroinflammation is a major risk factor in Parkinson's disease (PD). Alternative approaches are needed to treat inflammation, as anti-inflammatory drugs such as NSAIDs that inhibit cyclooxygenase-2 (COX-2) can produce devastating side effects, including heart attack and stroke. New therapeutic strategies that target factors downstream of COX-2, such as prostaglandin J2 (PGJ2), hold tremendous promise because they will not alter the homeostatic balance offered by COX-2 derived prostanoids. In the current studies, we report that repeated microinfusion of PGJ2 into the substantia nigra of non-transgenic mice, induces three stages of pathology that mimic the slow-onset cellular and behavioral pathology of PD: mild (one injection) when only motor deficits are detectable, intermediate (two injections) when neuronal and motor deficits as well as microglia activation are detectable, and severe (four injections) when dopaminergic neuronal loss is massive accompanied by microglia activation and motor deficits. Microglia activation was evaluated in vivo by positron emission tomography (PET) with [¹¹C](R)PK11195 to provide a regional estimation of brain inflammation. PACAP27 reduced dopaminergic neuronal loss and motor deficits induced by PGJ2, without preventing microglia activation. The latter could be problematic in that persistent microglia activation can exert long-term deleterious effects on neurons and behavior. In conclusion, this PGJ2-induced mouse model that mimics in part chronic inflammation, exhibits slow-onset PD-like pathology and is optimal for testing diagnostic tools such as PET, as well as therapies designed to target the integrated signaling across neurons and microglia, to fully benefit patients with PD.     

A suitable model for the utilization of Duddingtonia flagrans fungus in small-flock-size sheep farms

Effective alternatives to anthelmintic treatment of nematode parasite infections of sheep are required because of the high prevalence of drug resistance. Within this context, the nematode-trapping fungus Duddingtonia flagrans has become a valuable component of various integrated control strategies. Toward this objective, a small quantity of lyophilized D. flagrans chlamydospores (10⁶ spores per animal) was administered to sheep in a one-year plot study. Animals grazing on native pasture were divided into two homogeneous groups and were kept in 1-ha paddocks in the southern region of Brazil. The oral administration of chlamydospores led to a significant reduction (p < 0.05) in the number of nematode eggs per gram of feces and in the larval availability on herbage (difference of 37.6%) in comparison to the control group. Control animals needed to be dewormed three times during the experiment, whereas the fungus-treated animals maintained a low parasite load, independent of seasonal variation. Although D. flagrans cannot serve as a panacea for nematode parasite control of livestock, it represents a significant advance toward rationalizing the use of endoparasitic drugs in small animals.     

The pregnant sheep as a model for human pregnancy

Successful outcome of human pregnancy not only impacts the quality of infant life and well-being, but considerable evidence now suggests that what happens during fetal development may well impact health and well-being into adulthood. Consequently, a thorough understanding of the developmental events that occur between conception and delivery is needed. For obvious ethical reasons, many of the questions remaining about the progression of human pregnancy cannot be answered directly, necessitating the use of appropriate animal models. A variety of animal models exist for the study of both normal and compromised pregnancies, including laboratory rodents, non-human primates and domestic ruminants. While all of these animal models have merit, most suffer from the inability to repetitively sample from both the maternal and fetal side of the placenta, limiting their usefulness in the study of placental or fetal physiology under non-stressed in vivo conditions. No animal model truly recapitulates human pregnancy, yet the pregnant sheep has been used extensively to investigate maternal-fetal interactions. This is due in part to the ability to surgically place and maintain catheters in both the maternal and fetal vasculature, allowing repeated sampling from non-anesthetized pregnancies. Considerable insight has been gained on placental oxygen and nutrient transfer and utilization from use of pregnant sheep. These findings were often confirmed in human pregnancies once appropriate technologies became available. The purpose of this review is to provide an overview of human and sheep pregnancy, with emphasis placed on placental development and function as an organ of nutrient transfer.     

Duodenal ulcer induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)

Experiments in rats revealed that the parkinsonian drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) given in multiple daily doses either per os (p.o.) or subcutaneously (s.c.) induced in a dose-dependent manner solitary or double ("kissing") duodenal ulcers in the rat. MPTP also diminished cerebral concentrations of DOPAC and the duodenal ulcers were prevented by pretreatment with dopamine agonists (e.g., bromocriptine, lergotrile) or monoamine oxidase inhibitors (e.g., pargyline, 1-deprenyl). High doses of MPTP also caused gastric erosions and motility changes resembling parkinsonism (e.g., akinesia, rigidity, forward bending of trunk). This chemical decreased gastric secretion of acid and pepsin, as well as pancreatic bicarbonate, trypsin and amylase. Thus, MPTP causes duodenal ulcers that are possibly associated with impaired defense in the duodenal bulb (e.g., decreased availability of duodenal and pancreatic bicarbonate).