Mol Cell Biochem (2009) 328:183–188 DOI 10.1007/s11010-009-0088-3
Hippocampal neurodegeneration in experimental autoimmune encephalomyelitis (EAE): potential role of inflammation activated myeloperoxidase Mir Sajad Æ Jamil Zargan Æ Raman Chawla Æ Sadiq Umar Æ Mir Sadaqat Æ Haider A. Khan
Received: 12 January 2009 / Accepted: 11 March 2009 / Published online: 20 March 2009 Ó Springer Science+Business Media, LLC. 2009
Abstract Experimental Autoimmune Encephalomyelitis (EAE) is a well-established animal model of human multiple sclerosis (MS). The effect of this inflammatory disease on hippocampus has not been addressed. Keeping in view the above consideration an attempt was made to delineate the effect of EAE on the hippocampus of Wistar rats. The assessment of the damage to the hippocampus was done 16 days post induction by the immunolocalization of ChAT (choline acetyl transferase). ChAT decreased remarkably after induction that revealed cholinergic neuronal degeneration in the hippocampus. Subsequently, many biochemical parameters were assessed to ascertain inflammatory activation of nitric oxide and associated oxidative damage as a putative mechanism of the cholinergic degeneration. Nitric oxide metabolites increased significantly (P \ 0.05) with enhancement of MPO (Myeloperoxidase activity) (P \ 0.001) in the MOG (myelin oligodendrocyte protein) group as compared to the controls. Peroxidation of biomembranes increased (P \ 0.001), while reduced glutathione depleted (P \ 0.001) with parallel decrease in catalase (P \ 0.01) and superoxide dismutase enzyme activity (P \ 0.001) in the MOG group. Our results show a
M. Sajad J. Zargan S. Umar H. A. Khan (&) Developmental Toxicology Laboratory, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110062, India e-mail:
[email protected] R. Chawla Division of CBRN Defence, Institute of Nuclear Medicine and Allied Sciences (INMAS), New Delhi 110054, India M. Sadaqat Department of Internal Medicine, SKIMS Medical College Hospital, Bemina, Srinagar 190018, India
strong role of peroxidase dependent oxidation of nitrite and oxidative stress in cholinergic degeneration in EAE. Keywords Experimental autoimmune encephalomyelitis Hippocampus Immunolocalization Cognitive deficits Myeloperoxidase Nitric oxide
Introduction Inflammation is the hallmark of the demyelination in multiple sclerosis (MS). Enhanced cerebrovascular permeability with marked cellular infiltration especially monocyte derived macrophages and activation of microglia cause the onset of acute MS lesions [1–4]. These macrophages release many inflammatory mediators including cytokines that are contributing factors of the cell death in neurodegenerative disorders [5, 6]. Cognitive dysfunction affects about half of the individuals suffering from MS [7] and difficulties in learning and remembering new information represent the other most common cognitive deficits [8, 9]. Verbal memory deficits are observed in the progressive form of the disease and visuospatial memory deficits are observed in the relapsing– remitting form [10]. Hippocampus is archicortical cholinergic rich region and acts as a memory device [11] especially sensitive to various insults including inflammation [12]. Cellular death in this region can lead to depleted acetylcholine synthesis and can trigger memory deficits. Choline acetyl transferase is the most important enzyme in the acetylcholine synthesis as has been documented to be decreased in the progressive course of MS which is an indicator of cholinergic cell death [13]. Apart from this, hippocampal atrophy has also been documented in clinical investigations [14].
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From all the above evidences, it seems that the inflammatory events degenerate the cholinergic cells. The increased content of myeloperoxidase which is a potent catalyst for the oxidation of nitric oxide has been seen to increase in the cerebral cortex of MS tissue [15]. These activated oxidants like metabolites of nitric oxide (NO) may affect the integrity of the membranes and proteins and increase the oxidative stress which can lead to the cellular death and depletion of acetylcholine. This study is an attempt to investigate the peroxidase dependant oxidation of nitric oxide as an alternate mechanism to the exitotoxic neurodegeneration and has not been addressed in the EAE.
Materials and methods Chemicals Thiobarbituric acid (TBA), trichloroacetic acid (TCA), 5-50 dithio-bis-2-nitro benzoic acid (DTNB), Nitroblue tetrazolium(NBT), nicotinamide adenine dinucleotide(NADH), hexadecyltrimethylammonium bromide (HTAB),sodium guaiacol were purchased from SD Fine chemicals India. 3-30 Diaminobenzidine (DAB) with metal enhancer, anticholineacetyl transferase antibody (ChAT), anti-Rat IgG (whole molecule) peroxidase secondary antibody produced in rabbit, normal goat serum (NGS), freud’s complete adjuvant (CFA), and Griess Reagent system were purchased from Sigma Chemical Co. (St. Louis, USA). MOG (33–35) was purchased from Alexis Axxora USA, Aspergillus nitrate reductase from Boehringer Mannheim, Germany. All other routine chemicals used in this investigation were of research grade. Induction of EAE Young male Wistar rats (150 ± 10 g) were obtained from Jamia Hamdard central animal house after obtaining ethical committee clearance. Animals were housed (n = 2/cage) under a 12:12 h light–dark cycle which starting at 08.00 h after initial acclimatization for about 1 week, and permitted food and water ad libitum. Randomly each animal was assigned to two groups with 10 animals per group, group 1 received 100 ll of inoculum containing 50 lg MOG (myelin oligodendrocyte protein) in saline emulsified in CFA (Sigma St. Louis USA) (1:1) containing heat killed 1 mg Mycobacterium strain HR 37 a subcutaneous route at the base of the tail (now onwards MOG) group) and group 2 receiving CFA only (now onwards Adjuvant control group). Animals were scored daily for neurological signs (0, healthy; 1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb paralysis; 4, scale 3 plus forelimb weakness; and 5, moribund or dead).
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Immunohistochemistry of choline acetyl transferase (ChAT) Immunohistochemistry of hippocampal ChAT was performed as reported earlier [16]. Following anesthesia with sodium pentobarbital (40 mg/kg, i.p.), the rats were transcardinally perfused with 150 ml PBS (phosphate buffered saline) pH 7.4 followed by 200 ml of 4% chilled paraformaldehyde. After perfusion the brains were removed and prefixed in the above fixative (4% paraformaldehyde) overnight. The tissues were transferred to 30% sucrose in PBS (0.1 M) and kept overnight and subsequently sucrose solution was changed after 24 h for cryopreservation. Then 30 lm sections were cut through the coronal plane under frozen condition using microtome and incubated shortly in 0.3% H2O2 in methanol followed by incubation in blocking buffer containing 0.1 M PBS, 0.04% Triton X-100, and 10% NGS. Sections were incubated in anti-Rat anti-ChAT antiserum (Sigma) (1:500 dilutions) for 72 h at 4°C. After rinsing in buffer, sections were incubated in a anti-Rat IgG (whole molecule) peroxidase secondary antibody. The peroxides complex was visualized with 3,3-diaminobenzidine with metal enhancer in the presence of 0.024% hydrogen peroxide in PBS. Slides were prepared after putting the sections in mounting medium on a glass slide and dried. Photomicrography was done with the help of a microscope fitted with CCD camera (Nikon TES 2000U, Japan). Measurement of nitric oxide (NO) metabolites: Griess reaction After the experiment, animals were sacrificed and the brains were washed with PBS and placed on ice. Brains were dissected and hippocampal areas taken out by scalpel and forceps and quickly weighed and placed in borosilicate glass tubes containing chilled PBS, volume 10 times the tissue weight. Tissues were then homogenized on ice using a tissue homogenizer (Remi India). The homogenate was centrifuged at 100009g for 20 min. The supernatant was collected and centrifuged again at 750009g for 15 min. A total of 500 ll of supernatant was filtered through a micron filter (0.2 lm) and filtrate collected and immediately assayed as the refrigeration can degrade the NO metabolites. Briefly the 50 ll sample was incubated with Aspergillus nitrate reductase and cofactors at room temperature. After 90 min, equal amount of Griess reagent system was added and absorbance was read at 540 nm using a plate reader (Bio-Rad USA). Each experiment included a standard curve for nitrite and nitrate. Standard curves were used to determine NO metabolite concentration (lmol/l), which was divided by wet tissue weight and expressed as lmol/
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mg wet tissue weight. Nitrite contents of the sample (100 ll) was determined by reading absorbance after adding Griess reagents in the absence of nitrate reductase. Subtraction of nitrite values from the total NO metabolites allowed quantification of nitrate. All measurements were performed in triplicate.
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developed by addition of 0.5 ml of 1% w/v TBA dissolved in 0.05 N NaOH and kept in boiling water bath for 15 min until the appearance of pink color. The absorbance was read at 532 nm in a spectrophotometer. The result was expressed as nmoles MDA formed/min/mg protein. Reduced glutathione (GSH)
Myeloperoxidase activity (MPO) MPO activity was determined by the method as described earlier [17]. Small blocks of hippocampal tissue were finely minced at 4°C and homogenized with a homogenizer (Remi India) for 30 s in 10 mM potassium phosphate buffer (pH 6.0) with 0.5% hexadecyltrimethylammonium bromide in an approximate concentration of 50 mg of tissue/1 ml. This homogenate was sonicated for 30 s, and an aliquot (1.5 ml) was then frozen and thawed twice for the full release of MPO polymorphonuclear neutrophils. After centrifugation at 150009g for 15 min at 4°C, an aliquot (5–100 ll) of the final supernatant was mixed with 3 ml of 10 mM potassium phosphate buffer (pH 6.0) containing 0.07% of 20 mM aqueous guaiacol, and 20 ll of 3% hydrogen peroxide. Absorbance at 470 nm was read with a spectrophotometer. One unit of MPO activity was defined as the quantity catalyzing the decomposition of 1 lmol of hydrogen peroxide to water per minute at 37°C. Protein content was measured by Bradford method. MPO activity was expressed in units per mg protein.
GSH was measured in the groups following the method reported earlier [19]. Briefly, homogenized hippocampal tissue (10% w/v in phosphate buffer pH 7.4) was deproteinized by adding an equal volume of 10% TCA and was allowed to stand at 4°C for 2 h. The contents were centrifuged at 20009g for 15 min supernatant was added to 2 ml of 0.4 M Tris buffer (pH 8.9) containing 0.02 M EDTA (pH 8.9) followed by the addition of 0.01 M DTNB. Finally, the mixture was diluted with 0.5 ml of distilled water, to make the total mixture to 3 ml and absorbance was read in a spectrophotometer at 412 nm and results are expressed as lg GSH/g tissue. Total superoxide dismutases (SOD) activity
In order to assess free radical mediated effects following MOG injection estimations of lipid peroxidation (LPO) and reduced glutathione (GSH) were carried out with other enzymatic antioxidants (total superoxide dismutase and catalase) in hippocampus. Rats were sacrificed by cervical dislocation followed by decapitation and brains were dissected quickly on ice pack, removing the hippocampal tissues and weighed. The homogenates of the tissue were prepared in accordance with the test requirements as indicated therein.
Total SODs were measured in hippocampal region of the animals by an already described method [20]. In brief, 3 ml of assay mixture consisted of sodium pyrophosphate buffer 1.2 ml (0.082 M, pH 8.3), PMS 0.3 ml (186 lM), NBT 0.3 ml (300 lM), NADH 0.2 ml (780 lM), and 1 ml of 10% hippocampal tissue homogenate (prepared in 0.1 M phosphate buffer). The reaction was initiated by addition of NADH, followed by incubation at 37°C for 90 s. The reaction was stopped by addition of 1 ml glacial acetic acid and the reaction mixture was vigorously shaken with 4 ml of n-butanol. The mixture was allowed to stand for 10 min and centrifuged for 10 min at 15009g and butanol layer was separated. The color intensity of the formazan in butanol layer was measured at 560 nm against butanol using a spectrophotometer. A mixture without enzyme preparations was run in parallel, which served as blank. The SOD activity is expressed in nmol formazan formed/ min/mg protein.
TBARS, marker of lipid peroxidation (LPO)
Catalase activity
LPO in hippocampal areas was measured by estimating malonaldialdehyde (MDA) levels by thiobarbituric acid reactive substances (TBARS) as described already [18]. In brief, brain homogenate was prepared in 0.15 M KCl (5% w/v homogenate) and aliquots of 0.6 ml was incubated for 0 and 1 h at 37°C. Subsequently, 1.2 ml of 28% w/v TCA was added and the volume was made up to 3 ml by adding 1.2 ml of water. Following centrifugation at 30009g for 10 min, 2.5 ml of the supernatant was taken and color was
Catalase activity in the hippocampal tissues was assayed according to earlier reported method [21] using H2O2 as substrate. The reaction mixture of 1.5 ml consisted of 1 ml phosphate buffer (0.01 M, pH 7.0), 0.4 ml distilled water, and 0.1 ml of 10% homogenate (prepared in 0.1 M phosphate buffer). Reaction was started by adding 0.5 ml H2O2, incubated at 37°C for 1 min and reaction was stopped by addition of 2 ml of dichromate:acetic acid reagent (3:1). The tubes were immediately kept in a boiling water bath
Studies related to oxidative stress
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Control(Adj) EAE(MOG) 0.4
*
0.2
0.0
Results Choline acetyl transferase (ChAT) immunohistology The functional viability of ChAT neurons in the hippocampus was assessed by mapping the important enzyme of acetylcholine synthesis (choline acetyl transferase) using monoclonal antibody against ChAT. In MOG rats, number of surviving ChAT neurons was remarkably less (Fig. 1) as compared to those in the adjuvant control group (Fig. 1). NO metabolite levels Total NO metabolites in the Adjuvant control group was 0.286 ± 0.030 and that of the MOG treated group was 0.610 ± 0.034 increasing significantly (P \ 0.05). Moreover, the oxidized metabolites viz nitrate and nitrite were significantly (P \ 0.05) higher in the MOG group as compared to the adjuvant control group (Fig. 2). Fig. 1 ChAT immunoreactive neurons (arrows shown) in hippocampus of the Adjuvant controls and MOG treated rats (a and b, respectively). The ChAT immunoreactive neurons degenerated remarkably after subcutaneous injection of MOG (50 lg/100 ll inoculum). Original magnification 209
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N
itr
ite
e at it r
N
All the data presented were mean ± SEM. The variation within the groups was analyzed by unpaired one tailed t-test. In case of NO metabolites, the paired one tailed t-test was employed. Any variations with P \ 0.05 were considered to be significant.
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et
ab
ol
Statistical analysis
N
Protein content was determined by Bradford method 1976 [22].
*
0.6
s
Protein content
NO metabolites in hippocampus 0.8
ite
for 15 min and centrifuged for 10 min (15009g). The green color developed during the reaction was read at 570 nm in a spectrophotometer. Control tubes, devoid of enzyme, were also processed in parallel. The enzyme activity is expressed as nmol H2O2 consumed/min/mg protein.
Mol Cell Biochem (2009) 328:183–188 Total NO metabolites(µM/mg wet tissue)
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Fig. 2 Nitric oxide (NO) metabolites in the hippocampus of rat brain tissue filtrates from Adjuvant controls and MOG treated groups. Nitrate was calculated by subtracting nitrite values from total NO metabolite values. All the metabolites increased significantly in the MOG group (P \ 0.05) as compared to the control group. Values represent mean ± SEM of six rats. *Paired t-test, one tailed P \ 0.05
Myeloperoxidase (MPO) The concentration of MPO in adjuvant control group was estimated to be 7.000 ± 1.183 and that of the MOG group was 23.17 ± 3.070, revealing a significant (P \ 0.001) increase (Fig 3). LPO and GSH level A significant increase (P \ 0.001) in lipid peroxidation was observed in MOG group when compared to Adjuvant control group (Fig. 4). GSH suffered a significant decrease (P \ 0.001) (Fig. 4) in MOG treated rats. SOD and catalase activity A significant decrease (P \ 0.001) in SOD and catalase (P \ 0.01) activity was observed in hippocampus of MOG group when compared Adjuvant control group (Table 1).
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187 Table 1 Total superoxide dismutase (SOD) and catalase activity in hippocampus of adjuvant controls and MOG treated rats
***
Treatment groups
Total SOD activity (nmol formazan formed/min/mg protein)
Catalase activity nmol H2O2 consumed/ min/mg protein)
Adjuvant control (n = 6)
7.256 ± 0.004
7.735 ± 0.040
MOG (n = 6)
2.362 ± 0.012b
6.062 ± 0.311a
20
10
Significant decrease of SOD and catalase activity in MOG treated rats is evident as compared to adjuvant controls. Values represent mean ± SEM of six rats. Total SOD and Catalase activity 0
a
Unpaired, one tailed t-test, P \ 0.01
b
Unpaired, one tailed t-test, P \ 0.001
reported earlier to be elevated during the development of MS lesions [30]. Results in this investigation have demonstrated a myeloperoxidase driven activation of nitrite. Latter increased significantly (P \ 0.05) with other metabolites in the MOG rats that can be another possibility to above said in nitrosylation of proteins and is evident from the increased MPO (P \ 0.001) being consistent with the earlier findings where MPO levels have been shown to increase in MS tissue [15] and confirming the existence of the dependence of NO metabolite activation on myeloperoxidase as suggested separately [31]. This nitro stress works in conjunction with oxidative stress/environment, as shown by increased lipid peroxidation of biomembranes (P \ 0.001), reduced glutathione levels (P \ 0.001) with decrease in other enzymatic antioxidants viz superoxide dismutases (P \ 0.001) and catalase (P \ 0.01) in the hippocampus of MOG group may lead to the degeneration of cholinergic neurons as mapped by the immunoreactivity of ChAT (Fig. 1). The latter decreased significantly after MOG injection (50 lg) showing consistency with earlier reports [13]. The average neurological score in MOG group was three as compared to the 0 of Adjuvant controls paralleling the above results. The present study is an early attempt to understand the mechanisms of hippocampal neurodegeneration other than
C
Groups Fig. 3 Myeloperoxidase activity in hippocampus of adjuvant controls and MOG treated rats. MPO activity increased significantly (P \ 0.001) in the MOG treated rats. Values represent mean ± SEM of six rats. ***Unpaired t-test, one tailed P \ 0.001
Discussion
(a)
Lipid Peroxidation 25
*** µ g GSH/g tissue
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Fig. 4 Lipid peroxidation (LPO) (a) and reduced glutathione (GSH) level (b) in hippocampus of Adjuvant control and MOG treated rats. Significant increase of LPO and decrease in GSH level in MOG rats. Values represent mean ± SEM of six rats. Unpaired one tailed t-test, ***P \ 0.001. MOG versus adjuvant controls
nM MDA formed/min/mg protein
The evidences showed in this investigation highlight the role of peroxidase dependant activation of nitric oxide and oxidative stress as another possible mechanism for the degeneration of cholinergic neurons in MS. Until now many studies have revealed exitotoxic damage/apoptosis as the major cause of hippocampal degeneration [23–25]. In MS, inflammation causes disruption of the blood brain barrier [26], harboring of the macrophages and release of inflammatory oxidants as the earliest events. Nitric oxide is an important signaling molecule in central nervous system with diverse roles [27]. The elevated nitrite/nitrate ratio in CSF has been postulated to a biological indicator of the neurodegenerative phase of MS [28]. After induction of inflammation, the nitric oxide can react with superoxide forming peroxynitrite (ONOO-) [29] and promote the nitrotyrosine formation which has been
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MPO activity(Unit/mg protein)
Myeloperoxidase activity 30
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exitotoxicity in MS and opens a way for potential drug discovery. But the interactions of primary inflammation and target intracellular molecules need to be studied well with better methods to establish this degenerating mechanism.
Mol Cell Biochem (2009) 328:183–188
15.
16. Acknowledgments Authors are thankful to Dr. G. N. Qazi (Vice Chancellor, Jamia Hamdard) for continuous support during this study. Mir Sajad is recipient of Senior Research Fellowship (SRF) from Ministry of Health and Family Welfare, Government of India, New Delhi.
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