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Hematological disorders in 6-hydroxydopamine-induced rat model of Parkinson's disease/6-hidroksidopaminle induklenmis Parkinson hastaligi sican modelinde hematolojik bozukluklar.

Introduction

Parkinson's disease is a human neurodegenerative disorder primarily characterized by a massive and progressive degeneration of the dopaminergic neurons in the substantia nigra (SN). The most widely used animal models of Parkinson's disease involve intracranial infusion of the neurotoxin 6-hydroxydopamine (6-OHDA) directly into the ascending dopaminergic forebrain bundle, thereby inducing severe dopaminergic neuronal degeneration associated with profound deficits in feeding, drinking, and sensorimotor and learning functions [1-4]. Alternatively, new Parkinsonian rat models have been developed with 6-OHDA injected directly into the striatum to induce selective and moderate neurodegeneration of dopamine (DA) nerve terminals [5]. Similarly, in Parkinson's disease, the progressive degeneration of nigral dopaminergic neurons results in motor deficits only after 80% of the nigrostriatal system has degenerated [6]. It has been known that the brain can communicate with the immune system through either the hypothalamic pituitary (HP) axis or the sympathetic nervous system (SNS) [7,8]. The possible roles of these two major pathways in regulation of the hematopoiesis processes were examined by using pharmacological agents such as desipramine and 6-OHDA in order to determine their effects on the hematological parameters. It is also known that 6-OHDA is a useful neurotoxic agent that can reversibly impair the sympathetic nerve terminal [9,10]. When it is injected intravenously or intraperitoneally, it accumulates in the peripheral sympathetic nerve terminal and selectively destroys the sympathetic nerves. Its toxic effects directly result from its ability to generate free radical species, and from covalent bonding of quinone oxidant product [11]. With this chemical agent, we can create pure sympathectomized rats, which is reversible with the administration of desipramine, a competitive inhibitor of 6-OHDA [12]. Moreover, Bazan [13] reported the possible relationship between the nervous system and hematopoiesis. These results suggest that there is some erythropoietic regulation via the autonomic nervous system. However, the mechanism causing anemia associated with autonomic dysfunction is not well explained. Catecholamines and their corresponding receptors are widely distributed in both the central and peripheral nervous system. Besides their vasoactive effect [14], catecholamines have been known to be involved in different forms of learning and memory [4,15]. Norepinephrine (NE) particularly at the locus coeruleus (LC) area not only can regulate the hormone release from the HP axis but also the activity of the SNS [16]. Immune cell types associated with innate immunity such as natural killer cells, neutrophils, and macrophages are the potential subjects to be regulated by catecholamines because these cells express functional, [beta]2- and/or [alpha]- adrenergic receptors [17].

In summary, the primary goal of this study was to evaluate whether disordered hematopoiesis regulation via substantia nigra neuron lesion may induce hematological disorders.

Materials and Methods

Animals

Thirty male Wistar rats weighing 200 [+ or -] 50 g at the start of the experiment were used. The animals were housed in a temperature- and light-controlled room (22[degrees]C, 12-h cycle starting at 08:00 h) and were fed and allowed to drink water ad libitum. Rats were treated in accordance with the guidelines of Animal Bioethics from the Act on Animal Experimentation and Animal Health and Welfare Act from Romania, and all procedures were in compliance with the European Council Directive of 24 November 1986 (86/609/EEC).

Neurosurgery and Drug Administration

The rats were anesthetized with sodium pentobarbital (45 mg/kg b.w. i.p., Sigma). Right-unilateral lesioning of the substantia nigra was performed by stereotaxic microinjections of 8 micrograms (free base) 6-OHDA, dissolved in 4 [micro]l physiological saline containing 0.1% ascorbic acid, administered through the Hamilton microsyringe over 4.5 minutes. The syringe was left in place for 5 minutes after injection before being slowly removed. The rats were pretreated 30 minutes before the 6-OHDA infusion with 25 mg/kg intraperitoneal desipramine (Sigma) to protect noradrenergic projections. Sham-operated rats received an injection of desipramine, followed by vehicle only in the substantia nigra. The following coordinates were used: 5.5 mm posterior to bregma; 2.0 mm lateral to the midline; and 7.4 mm ventral to the surface of the cortex [18]. Hematological parameters were assayed one week after the neurosurgery.

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Blood Sampling Protocol

One week after neurosurgery, blood samples were withdrawn via the Biotrol sampling catheter from 15 sham-operated and 15 6-OHDA-treated rats. Blood samples (0.5 ml approximately/sample) were collected in vials containing EDTA for hematological investigations.

Hematological parameters were assayed by a COULTER[R] Ac x T 5diff[TM] CP-precision instrument for hematology research.

Histological Control

At the end of the experiment, all rats were sacrificed with an overdose of sodium pentobarbital (100 mg/kg i.p.) followed by a transcardial infusion of 0.9% saline and a 10% formalin solution. The brains were removed and placed in a 30% sucrose/ formalin solution. The brains were frozen and cut into coronal sections (50 [micro]m) using a freezing microtome and stained with cresyl violet for verification of the point of the syringe needle. Only experimental data from lesions correctly located in the substantia nigra were used for statistical analysis.

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Data Presentations and Statistical Analysis

Results were expressed as mean [+ or -] S.E.M. Because the data were not normally distributed, the non-parametric statistic Mann-Whitney U test was employed. Results were considered significant if p<0.05. The number of observation was 30.

Results

Experimental data were registered one week after the 6-OHDA administration. In the 6-OHDA-lesioned rats, we observed a significant decrease in the total number of white blood cells [6.16 [+ or -] 0.3 [10.sup.3]/[mm.sup.3] vs 6.8 [+ or -] 0.4 [10.sup.3]/[mm.sup.3], U=36.5 p<0.03 - neutrophils (0.9 [+ or -] 0.2 vs 0.8 [+ or -] 0.2 [10.sup.3]/[mm.sup.3]), lymphocytes (4.5 [+ or -] 0.8 vs 5.6 [+ or -] 1.7 [10.sup.3]/[mm.sup.3])] (Figure 1), red blood cells (5.01 [+ or -] 0.1 106/[mm.sup.3] vs 5.8 [+ or -] 0.2 106/[mm.sup.3], U=50.5 p<0.01) (Figure 2), and hemoglobin level (8.9 [+ or -] 0.2 g/L U=50.5 vs 9.9 [+ or -] 0.3 g/L, p<0.02) (Figure 3) compared with the sham-operated groups. 6-OHDA significantly increased mean cell volume (MCV) (51.8 [+ or -] 0.8 fL vs 50 [+ or -] 0.5 fL, U=28.5 p<0.04) and mean cell hemoglobin (MCH) (17.8 [+ or -] 0.1 pg vs 17.08 [+ or -] 0.2 fL, U=26.5 p<0.01) compared with sham-operated groups (Figures 4, 5).

[FIGURE 5 OMITTED]

Discussion

It is well recognized that the immune response is under the influence of a variety of neural or neuroendocrine mechanisms. Much less studied is the possible influence of these mechanisms on hematopoiesis.

In our previous studies, we reported that the central dopaminergic system has a crucial role in regulation of the immune processes as well as hematopoiesis [19,20]. In our present study, we used a procedure of chemical sympathectomy by lesioning the substantia nigra with 6-OHDA. By means of this particularly electrolytic lesion, we observed a significant decrease in hematological parameters registered one week after 6-OHDA administration, tested by the total number of leukocytes, erythrocytes, hemoglobin level and the erythrocyte indexes (MCV and MCH). We demonstrated that rats treated with 6-OHDA showed anemia. In addition, since the WBC significantly decreased during 6-OHDA-induced anemia, the effect of 6-OHDA in this experiment may be specific for erythropoiesis, as well as for bone morrow suppression.

The regulation of the hematopoietic system is achieved at three levels: 1) at the cellular level of bone marrow stroma, 2) at the humoral level by cytokines, and 3) by catecholamines and other neuroendocrine factors.

Sympathetic nerve endings and bone marrow cells are the main source of bone marrow catecholamines [21,22]. Among the catecholamines, a substantial amount of dopamine was detected in bone marrow [23]. Bone marrow catecholamines originate from sympathetic nerve fibers and from hematopoietic cells directly. Catecholamines of neural origin show a circadian rhythmicity. Adrenoceptors present on bone marrow cells include the 1-subtype, which seems to mediate the catecholaminergic control of hematopoiesis. It has been well documented that there are [alpha]-and, [beta]-adrenergic receptors on the surface of the erythrocytes. However, [alpha]-receptors are more important in erythropoiesis. Administration of an [beta]-adrenergic stimulant did not elicit the erythropoietic effect, whereas a, [beta]-adrenergic stimulant induced erythropoiesis in an in vitro culture of erythroid progenitor cells [24]. Since, [beta]-adrenergic receptors are abundant on the surface of erythrocytes, the effect of, [alpha]-adrenergic blocking agents has often been investigated using erythrocytes [25,26].

In accordance with these findings, in our present study we observed some abnormalities of hematopoiesis after electrolytic lesion of the central dopaminergic neurons from the substantia nigra by means of 6-OHDA. 6-OHDA is a useful chemical agent for inducing neurogenic anemia.

Acknowledgements

This work was supported by a grant from the National Council of Scientific Research and University Education (grant CNCSIS, A type, no. 639/2006), Romania.

Received: October 21, 2007 Accepted: March 18, 2008

Gelis tarihi: 21 Ekim 2007 Kabul tarihi: 18 Mart 2008

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Lucian Hritcu

Department of Physiology, Alexandru Ioan Cuza University, Iasi, Romania

Address for Correspondence: Dr.Lucian Hritcu, Department of Physiology, Alexandru Ioan Cuza University, Iasi, Romania

Phone: +40-232201666 E-mail: hritcu@uaic.ro
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Title Annotation:Research Article
Author:Hritcu, Lucian
Publication:Turkish Journal of Hematology
Article Type:Report
Geographic Code:4EXRO
Date:Sep 1, 2008
Words:2259
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