NGF in experimental models of parkinson disease
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9 1996 by Humana Press Inc. All rights of any nature whatsoever reserved. 1044-7393/96/2801-03--0225 $06.00
NGF in Experimental Models of Parkinson Disease L. LORIGADOS,*P. ALVAREZ,N. PAVON, T. SERRANO,L. BLANCO,AND R. MACiAS Centro Internacional de Restauraci6n Neurol6gica, Ave. 2 5 No 1 5 8 0 5 e~ 1 5 8 y 160, Playa, C. Habana, Cuba Received April 7, 1995; Accepted July 20, 1995
ABSTRACT We have now applied the enzyme immunoassay using anti-NGF monoclonal antibody (MAb) 27/21 and a blocking test validating the specificity of the immunoreactivity for NGF in serum samples to examine NGF levels in normal rat sera, hemiparkinsonian rat sera, normal monkey sera, and MPTP-treated monkey sera. The levels of NGF in treated animals showed reductions when compared with serum from normal animals. The NGF level alterations observed in lesioned animals and in human parkinsonian patients evidence a relationship between this neurotrophic factor and the neurodegenerative changes observed in Parkinson disease (PD). Index Entries: NGF; serum; hemiparkinsonian rat; MPTP-treated monkey; Parkinson disease.
INTRODUCTION The experimental m o d e l s of p a r k i n s o n i s m , n o n h u m a n primates a n d rodents have b e e n extensively e m p l o y e d to assess ways to reverse the d o p a m i n e deficiency by different m e a n s of replacement therapy. Recently, we have reported low levels of NGF in s e r u m from patients with parkinson disease (PD) using a two-site e n z y m e i m m u n o a s s a y (EIA) (Lorigados "Author to whom all correspondence and reprint requests should be addressed. Molecularand ChemicalNeuropathology
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et al., 1992). Clearly, the application of a sensitive system for the detection of NGF in serum from animal models of parkinsonism would have a significant impact on our possibility to evaluate the involvement or therapeutic use of NGF in PD.
MATERIALS AND METHODS
Dopamlne Denervation Procedure Male Sprague-Dawley rats (200-250 g) were unilaterally lesioned under chloral hydrate anesthesia in the ascending median forebrain bundle of monoamine neurons (coordinates A-4.9; L 1.7; V 8.1) using 6-hydroxydopamine HCI (6-OH-DA [Sigma]; 8 mg/mL saline vehicle containing 0.1% ascorbate) (Marshall and Ungerstedt, 1977).
MPTP Treatment Male adults (12-15 yr) Macaca arctoides monkeys were anesthetized with ketamine (10 mg/kg) and then, via direct right internal carotid artery puncture, infused with 0.4 mg/kg MPTP at a rate of 4 mL/min to produce a left hemiparkinsonian syndrome (Bankiewicz et al., 1986).
Blood Samples Serum samples were collected from 15 experimental animals: 11 hemiparkinsonian rat sera (HPRS) and 4 MPTP-treated monkey sera (MPTP-MS). The experimental sera were compared with normal animal samples: 17 normal rat sera (NRS) and 16 normal monkey sera (NMS).
EIA for N G F A two-site EIA for NGF (SOderstrOm et al., 1990) and a blocking test validating the specificity of the immunoreactivity for NGF in serum described previously (Lorigados et al., 1992) were used.
RESULTS
Application of the NGF EIA to Sera from Hemiparkinsonian Rat and MPTP-Treated Monkeys fiera from hemiparkinsonian rats and MPTP-treated monkeys were tested (Fig. 1). There was, in all cases, a trend toward lower values of NGF detected in the animal sera. In MPTP-MS, the reductions were not statistically significant (Fig. 1A). However, the HPRS showed strikingly lower levels of NGF immunoreactivity in the EIA (Fig. 1B). Molecular and Chemical Neuropathology
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Fig. 1. Comparison of NGF levels among sera from animal models of parkinsonism (MPTP-MS and HPRS) and normal sera (NMS and NRS). (A) The result of MPTP-MS and NMS. (B) Levels of NGF from HPRS and NRS. In both cases, the serum shows reduced NGF levels compared with normal serum; in HPRS, the reduction was statistically significant. Statistical analysis was by Student's t-test (**p < 0.001).
DISCUSSION There is an increasing body of information concerning the physiological and potential pharmacological function of NGF in the peripheral and central nervous system. Although NGF is the best-characterized neurotrophic factor so far, little is k n o w n about its role in the pathogenesis of any disease. The experimental models of parkinsonism, n o n h u m a n primates and rodents, have been extensively e m p l o y e d to assess ways to reverse the d o p a m i n e deficiency by different means of replacement therapy. In order to clarify the physiological significance of serum NGF, we examined w h e t h e r the serum levels of NGF were correlated with certain neurological disorders. The EIA used in this work can detect small changes in NGF concentration that may occur u n d e r physiological and pathological conditions, such as PD. Previously, we reported low levels of NGF in serum from patients with PD (Lorigados et al., 1992). One question immediately comes to mind: H o w are the levels of NGF in serum from animals models of parkinsonism to be detected? Using the EIA antibody 27/21, it was possible to detect levels of immunoreactivity to NGF in NMS (0.183 + 0.05 ng/mL) and NRS (0.271 + 0.07 ng/mL). These levels were compared with the levels of NGF in experimental models of parkinsonism and the results s h o w e d reduction of NGF in hemiparkinsonian animals (Fig. 1). The NGF level alterations s h o w n in serum from these experimental models and h u m a n parkinsonian patients evidence a relationship between NGF and the neurodegenerative changes observed in PD.
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REFERENCES Bankiewicz K. S., Oldfield E. H., Chiueh C. C., Doppman J. L., Jacobowitz D. M., and Kopin I. J. (1986) Hemiparkinsonism in monkeys after unilateral internal carotid artery infusion of 1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP). Life Sci. 39, 7-16. Lorigados L., Si3derstri3m S., and Ebendal T. (1992) Two-site enzyme immunoassay for flNGF applied to human patient sera. J. Neurosci. Res. 32, 329-339. Marshall J. F. and Ungerstedt U. (1977) Supersensitivity to apomorphine following destruction of the ascending dopamine neurons: quantification using the rotational model. Eur. ]. Pharmacol. 41, 361-367. Si3derstri3m S., Hallbook F., Ibahez C. F., Persson H., and Ebendal T. (1990) Recombinant human f~-nerve growth factor (NGF): Biological activity and properties in an enzyme immunoassay. J. Neurosci. Res. 27, 665-677.
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