Rotenone induced neurotoxicity in rat brain areas: A study on neuronal and neuronal supportive cells
Supriya Swarnkar
a, Poonam Goswami
a, Pradeep Kumar Kamat
b, Ishan K. Patro
c, Sarika Singh
a, Chandishwar Nath
a*aDivision of Toxicology, CSIR-Central Drug Research Institute, Lucknow-226001 (U.P.), India
bDivision of Pharmacology, CSIR-Central Drug Research Institute, Lucknow-226001 (U.P.), India
cSchool of Studies in Neuroscience, Jiwaji University, Gwalior-474011 (M.P.), India
*Corresponding Author: Dr Chandishwar Nath, Chief Scientist & Head , Division of Toxicology, CSIR-Central Drug Research Institute, P.O.Box 173, Lucknow-226001, India, Fax: +91-522-2623405, Tel.: +915222212411-18- 4434, E-mail: [email protected]
Abstract
The present study was conducted to correlate rotenone-induced neurotoxicity with cellular and molecular modifications in neuronal and neuronal supportive cell in rat brain regions. Rotenone was administered (3, 6 and 12µg/µl) intranigrally in adult male SD rats. After 7th day of rotenone treatment, specific protein markers for neuronal cells-TH (tyrosine hydroxylase), astroglial cells- GFAP (glial fibrillary acidic protein), microglial- CD11b/c, and Iba-1 were evaluated by immunoblotting and immunofluorescenece in striatum (STR) and mid brain (MB). Apoptotic cell death was assessed by Caspase-3 gene expression. Higher doses of rotenone significantly lowered TH protein level and elevated Iba-1 level in MB. All the doses of rotenone significantly increased GFAP and CD11b/c protein in the MB. In STR, rotenone elevated GFAP level but did not affect TH, CD11b/c and Iba-1 protein level. Caspase-3 expression was increased significantly by all the doses of rotenone in MB but in STR only by higher doses (6 and 12µg). It may be suggested that astroglial activation and apoptosis play important role in rotenone-induced neurotoxicity. MB appeared as more sensitive than STR towards rotenone induced cell toxicity.
The astroglial cells emerged as more susceptible than neuronal and microglial cells to rotenone in STR.
Keywords: Rotenone, pesticide, neuronal cells, neuronal supportive cells and GFAP (glial fibrillary acidic protein)
Abbreviations: CNS, Central nervous system; STR, striatum; MB, mid brain; SN, substantia nigra; BSA, Bovine serum albumin; EDTA, ethylenediamine tetraacetic acid; PBS, phosphate buffer saline; ROS, reactive oxygen species; TH, Tyrosine hydroxylase; GFAP-ir, Glial fibrillary acidic protein immunoreactivity.
Introduction
Rotenone is the most potent member of the rotenoids, a family of isoflavonoids extracted from Leguminosae plants and used as organic pesticide. Being highly lipophilic, it freely crosses cell membrane, blood brain barrier and causes neurotoxicity by inhibiton of complex I of the mitochondrial electron transport chain (Talpade et al. 2000).
Neuronal supportive cells (astrocytes and microglia) have been implicated in neurodegeneration. Activation of astrocytes is seen in CNS pathologies, such as stroke, trauma, growth of a tumor, or neurodegenerative disease such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Simonian and Coyle, 1996). An increase in reactive microglial cells was found in the striatum and substantia nigra of patients with idiopathic PD (Beal 1996).
Rotenone exposure produces the selective and extensive microglial activation in the nigrostriatal pathway with minimal reactive astrocytosis in rats (Sherer et al. 2003a). Chronic treatment with rotenone significantly enhances immunoreactivity of both GFAP (a marker of reactive astrocytes) positive astrocytes and OX-6 positive microglial cells in aged groups as compared to young rats (Phiney et al. 2006). Rotenone activates microglial cells through ROS production, contributing to oxidative damage in selective brain areas (Sherer et al. 2002). It was reported that rotenone is able to alter astrocyte population in primary mesencephalic culture (Radad et al. 2008).
Rotenone induced neuronal death in spinal cord was accompanied by abundant astrogliosis and microgliosis as evident from GFAP and OX-42 immunoreactivity, respectively (Samantrey et al. 2007). Rotenone induced cell death in rat dopaminergic cell line is linked to mitochondrial complex I dysfunction (Marella et al. 2007).
Our earlier study with rotenone treatment in brain homogenates demonstrated that oxidative stress plays a key role in neuronal damage, which varies among the rat brain areas (Swarnkar et al. 2009). A correlation was also found among rotenone induced biochemical changes and cerebral damage in brain areas with neuromuscular coordination
tested by rota rod in rats (Swarnkar et al. 2010). Rotenone induces oxidative stress and apoptosis in Neuro-2a cells that is associated more with intracellular calcium rather than free radicals (Swarnkar et al. 2012a).
Earlier studies done with rotenone were chiefly based on predicting role of caspase mediated apoptosis in cell culture. However, the studies on comparative susceptibility of neuronal and neuronal supportive cells to rotenone in brain regions are scanty. Therefore, the present study was undertaken to investigate the cellular and molecular changes in neuronal and neuronal supportive cells along with apoptotic cell death caused by rotenone in STR and MB.
Materials and Method Materials
The chemicals rotenone, DMSO (dimethyl sulphoxide), chloral hydrate, sodium chloride (NaCl), potassium chloride (KCl), Tris–HCl, EDTA, phenylmethylsulfonyle fluoride (PMSF), pepstatin, protease inhibitor cocktail, bovine serum albumin (BSA), paraformaldehyde (PFA), rabbit anti TH (Tyrosine hydroxylase) antibody, rabbit anti GFAP antibody, mouse β-actin antibody and anti mouse HRP secondary antibody were procured from Sigma Chemicals Co. (St. Louis, MA, USA). Mouse anti Iba-1 antibody was purchased from Santa Cruz (CA, USA). Rabbit anti CD11b/c antibody was obtained from Abcam (Cambridge, MA, USA). Secondary antibodies anti-rabbit alexa 594 and anti-mouse alexa fluor 488 were purchased from Invitrogen. Anesthetic ether, SDS, Glycine, methanol and folin reagent of Sisco Research Laboratories Limited, Mumbai India were used in the study. Omniscript RT kit was procured from Qiagen, USA
Animals
The experiments were carried out with adult male Sprague–Dawley rats (180 -200g). The animals were kept in Polyacrylic cage and maintained under standard housing conditions (room temperature 22 ± 1oC and humidity 60- 65%) with 12h light and dark cycle (lights on at 6:00 a.m.). The food in form of dry chow pellets and water were available ad libitum. The animals were procured from the Laboratory Animal service Division of Central Drug Research Institute and experiments were approved by Animal Ethics committee of Central Drug Research Institute, Lucknow, India and performed according to internationally followed ethical standards.
Stereotaxic surgery
Rat was anesthetized with chloral hydrate (300 mg/kg, i.p.) and placed on a stereotaxic frame (Stoelting,USA), for surgery. Rotenone was dissolved in DMSO and infused (5μl) into the right SN. Similarly, vehicle (DMSO) was administered (5μl) into right SN in a separate group and this group was used for the comparison with rotenone treated groups. The stereotaxic coordinates for SN are AP: 5.3mm; L: 2.0mm; DV: 7.8mm, from the Bregma point (Paxinos et al., 1988). The needle was kept in position for about 5 min to prevent outflow. After the injection, burr hole was sealed with bone wax and antiseptic powder sprayed afterwards skin was sutured. The animals were kept in a cage for recovery with free access to food and water. The proper postoperative care was done by monitoring animals till they recover completely. Animals were divided in five groups viz. control (without any treatment), vehicle (DMSO), 3μg rotenone, 6μg rotenone and 12μg rotenone. Animals were sacrificed after 7th day of rotenone treatment and brain was isolated, dissected and processed as per assay requirement.
Tissue collection for western blotting
The rats perfused through heart with ice-cold normal saline under mild ether anesthesia. Brain were collected and kept over a glass plate placed on ice for 15 min and then dissected into different brain regions - Striatum (STR), Mid Brain (MB), according to Glowinski and Iversen (1966).
Western blotting in brain tissue samples
Brain tissues were collected and sonicated in suspension buffer containing 100mM NaCl, 10mM Tris–HCl, 1mM EDTA, 100μg/ml phenylmethylsulfonyle fluoride (PMSF), 0.2mg/ml pepstatin, and 5%w/v protease inhibitor cocktail and then centrifuged at 10,000g X 20 min. X 4°C. The supernatant was collected and measured protein (100μg) loaded on 10% polyacrylamide resolving gel overlaid with a 4% stacking gel (Laemmli, 1970).
Electrophoresed proteins were transferred electrophoretically onto Hyperfilm ECL (Amersham biosciences) membrane by the method of (Towbin, 1979), after which blots were blocked in 5% w/v BSA in TBS-T (20 mM Tris, 0.9% NaCl, 0.1% Tween-20, pH: 7.6) at 4°C overnight. Membrane was incubated with rabbit anti-TH (1:
1000) and rabbit anti-GFAP (1: 1000), and β-actin (1: 1000) polyclonal antibody for 2 hrs. at 37°C. While, membrane was incubated over night (20 hrs.) with primary mouse monoclonal Iba-1(1: 500) and mouse Integrin αM (CD11b/c) (1: 500) antibody at 4°C. Following washing with TBS-T for 1 hr at room temperature, membrane was incubated again with secondary antibody conjugated with horseradish peroxidase (1:2000 and 1:1000) at room
temperature for 1 hr. as per requirement. Later membrane was developed by chemiluminescence detection using ECL-Plus detection system (Pierce Biosciences) according to the instructions provided by manufacturer. Intensity of signals was captured onto an X-ray film (Hyperfilm, Amersham Biosciences). Relative optical intensity of bands was analyzed by using Alpha Image gel documentation software. Comparison between different treatment groups was done by determination of the examinated protein/ β-actin protein ratio of the immunoreactive area by densitometry.
Tissue processing and sectioning for immunofluorescence
Rats were perfused intracardially with ice-cold 0.1 M phosphate-buffered saline (PBS) followed by cold paraformaldehyde (4% wt/vol) in 0.1 M PBS. Animals decapitated and the brains were removed and processed for paraffin embedded sectioning. Sections collected on slides coated with poly-L-lysine and allowed to dry completely before use. Sequential sections (5µm thickness) of striatal region and mid brain {containing SN (substantia nigra)}
were cut by microtome (Leica RM 2255, Lab India). Brain regions were selected based on George Paxinos brain atlas [12]. The coordinates for these areas from the rostral to the caudal portion of the brain were:
(i) Striatal region taken in between 0.70 to 1.70 mm anterior to bregma and (ii) Mid-brain was taken in between -2.30 to -6.04 mm posterior to bregma.
Immunofluorescence (IF) in paraffin embedded sections
Deparaffinization of sections with two washes of xylene 15 minutes each was followed by rehydration of sections with 100%, 90%, 70%, 50% and 30% alcohol 3-3 minutes each. Rehydrated slides then washed twice with 1X PBS (1.3M NaCl, 70mM Na2HPO4, 30mM NaH2PO4) for 15 min. Sections were permeabilized with 0.5% Triton X-100 prepared in PBS, for 10 min. Sections were blocked for 2hrs in 5% BSA followed by incubation for 24 hrs with primary antibody TH; 1:500, GFAP; 1:500, Iba-1; 1:500) and mouse Integrin αM (CD11b/c) (1:200). After washing with PBS pH 7.4, the sections were incubated for 1hr in anti-rabbit alexa 594 (1:100) and anti- mouse alexa fluor 488 (1:100) respectively. Sections were washed in PBS and mounted by antifade and observed using fluorescent microscope (Nikon E200).
Immunofluorescence images were captured at 400× magnification by Fluorescent microscope (Nikon Eclipse E200, Japan) equipped with a super high pressure mercury lamp power supply (Nikon, Japan).
mRNA expression by reverse transcription PCR
RNA was isolated from brain using TRIzol reagent (Invitrogen) as per instructions provided by the manufacturers.
Concentration and purity of RNA were determined spectrophotometrically using Genequant. Approximately 2μg of total RNA was reverse transcribed using reverse transcriptase (RT) in a 20μl mixture containing oligo-(dT)-primer, RNase Inhibitor, dNTP mix and 5X reaction buffer (Omniscript RT kit).
The resultant cDNA was amplified separately with specific primer for Caspase-3 and β-actin using Taq PCR core Kit (Qiagen, USA). Briefly, cDNA was amplified in a 20μl reaction volume containing 1U Taq polymerase, 200μM (each) dNTP mix and 2μl 10 X Taq buffer with specific primers. The polymerase chain reaction mixture was amplified in a DNA thermal cycler (Bioer XP cycler) through 35-cycles at the specifications described in Table-1.
The PCR products were detected by electrophoresis on a 1.2 % agarose gel containing ethidium bromide.
Band intensities were quantified by computerized densitometry (Alpha Imager gel documentation system) and normalized with respect to β-actin mRNA.
Estimation of protein content
A protein concentration was estimated using bovine serum albumin (BSA) 0.01–0.1 mg/ml as standard according to the method of (Lowry et al. 1951).
Statistical analysis
The data was analyzed by one way analysis of variance (ANOVA) followed by post hoc Newman Keuls test.
Results are expressed as the Means ± SEM, value of P < 0.05 was considered as level of significance.
Results
TH immunoblotting
TH immunoblotting was performed to assess damage of neuronal cells. Rotenone 3, 6 and 12µg doses resulted in TH protein level 1.30±0.25, 1.06±0.19 and 1.04±0.22 respectively as compared to vehicle (1.69±0.35) in STR (F=1.039, df=4, p=0.4034; n=7). While in MB TH level was 0.59±0.13, 0.49±0.07 and 0.3622±0.043 by rotenone 3, 6 and 12µg doses respectively as compared to vehicle (0.96±0.09). But significantly lowered TH expression only by
6 and 12µg doses in MB (F=6.423, df=4, p=0.0007; n=7) Fig 1(a) and 1(b). The decrement in TH expression indicates loss of neuronal cells.
Fig. 1 TH expression in rat brain regions [7 days after rotenone (3, 6 and 12μg) treatment] (a) X- ray blot representative 1(b) Graphical representation showing the altered TH expression. Values are expressed as Means ± S.E.M and analyzed by ANOVA post hoc Newman-Keuls Multiple Comparison Test (n=7).
**P<0.001 and * P <0.05 Vehicle (DMSO) vs Rotenone treated group.
GFAP immunoblotting
GFAP immunoblotting was performed to assess expression of GFAP, which is a phenotypic marker for astroglial cells. All the doses of rotenone 3, 6 and 12µg caused significantly enhanced GFAP protein level 0.78±0.14, 0.91±0.20 and 1.07±0.27 respectively as compared to vehicle (0.23±0.033) in MB (F=5.958, df=4, p=0.0012; n=7).
Rotenone 3, 6 and 12µg doses resulted in GFAP protein level 0.36±0.05, 0.57±0.11 and 0.70±0.15 respectively as compared to vehicle (0.20±0.026) in STR (F=6.366, df=4, p=0.0008; n=7) but significant increase in STR only by higher doses 6 and 12µg. Fig. 2 (a) and 2(b). The increment in GFAP expression indicates astroglial activation.
Fig. 2 GFAP expression in rat brain regions [7 days after rotenone (3, 6 and 12μg) treatment] (a) X- ray blot representative 2(b) Graphical representation showing the altered GFAP expression in STR and MB. Values are expressed as Means ± S.E.M and analyzed by ANOVA post hoc Newman-Keuls Multiple Comparison Test (n=7). **P<0.001 and * P <0.05 Vehicle (DMSO) vs Rotenone treated group.
CD11b/c immunoblotting
CD11b/c immunoblotting was performed to assess expression of CD11b/c, which is a marker for microglial cells.
All the doses of rotenone 3, 6 and 12µg produced significantly elevated, i.e. 0.88±0.13, 1.03±0.19 and 1.21±0.24 CD11b/c protein level respectively as compared to vehicle (0.32±0.11) in MB (F=6.558, df=4, p=0.0006; n=7).
Rotenone 3, 6 and 12µg doses resulted in CD11b/c protein level 0.49±0.18, 0.58±0.23 and 0.65±0.24 respectively as compared to vehicle (0.41±0.13) in STR (F=0.5078, df=4, p=0.7303; n=7) but no significant change in STR. Fig. 3 (a) and (b). The increased expression of CD11b/c indicates microglial activation.
Fig. 3 CD11b/c expression in rat brain regions [7 days after rotenone (3, 6 and 12μg) treatment] (a) X- ray blot representative 3(b) Graphical representation showing the altered CD11b/c expression in STR and MB.
Values are expressed as Means ± S.E.M and analyzed by ANOVA post hoc Newman-Keuls Multiple Comparison Test (n=7). **P<0.001 and * P <0.05 Vehicle (DMSO) vs Rotenone treated group.
Iba-1 immunoblotting
Iba-1 immunoblotting was performed to assess microglial activation in brain. Rotenone 3, 6 and 12µg doses caused Iba-1 protein level 0.19±0.02, 0.18±0.02 and 0.19±0.01 respectively as compared to vehicle (0.17±0.01) in STR (F=0.6302, df=4, p=0.6448; n=7). While in MB Iba-1 level was 0.56±0.12, 0.84±0.19 and 1.33±0.29 by rotenone 3, 6 and 12µg doses respectively as compared to vehicle (0.25±0.05). However, significantly elevated Iba-1 expression only by 6 and 12µg doses in MB (F=8.142, df=4, p=0.0001; n=7) Fig 4(a) and (b). The increased expression of Iba-1 indicates microglial activation.
Fig. 4 Iba-1 expression in rat brain regions [7 days after rotenone (3, 6 and 12μg) treatment] (a) X- ray blot representative 4(b) Graphical representation showing the altered Iba-1 expression in STR and MB. Values are expressed as Means ± S.E.M and analyzed by ANOVA post hoc Newman-Keuls Multiple Comparison Test (n=7). **P<0.001 and * P <0.05 Vehicle (DMSO) vs Rotenone treated group.
Immunofluorescence (IF)
Immunofluorescence study was performed to assess the morphological expression of specific protein markers for neuronal cells-TH (tyrosine hydroxylase), astroglial cells- GFAP (glial fibrillary acidic protein), microglial- CD11b/c, and Iba-1 in STR (Image-1) and SN (Image-2).
Image:1 Immunofluorescence images [after 7 days of rotenone (3, 6 and 12μg) treatment in STR] the different marker proteins for neuronal and neuronal supportive cells (white arrows) and star shaped astrocytes (yellow arrow heads) bar=50µm.
Image:2 Immunofluorescence images [7 days after rotenone (3, 6 and 12μg) treatment in MB containing SN]
the different marker proteins for neuronal and neuronal supportive cells (white arrows) and star shaped astrocytes (yellow arrow heads) bar=50µm.
TH immunofluorescence
TH immunoreactivity (ir) assessed the morphological changes in neuronal cells. Rotenone 3, 6 and 12µg doses resulted in THir level in terms of % Area 49.76±2.86, 46.54±3.26 and 43.00±6.85 respectively as compared to vehicle (59.45±2.02) in STR (F=3.122, df=4, p=0.0469; n=4). THir level in STR (F=0.5354, df=4, p=0.7119; n=4) in terms of Count/Area by rotenone 3, 6 and 12µg doses was 2081.57±332.46, 2025.58±363.55 and 1577.12±314.44 respectively as compared to vehicle (2180.56±61.50). While THir level in terms of % Area was 28.19±5.01, 17.91±2.60 and 12.03±2.47 by rotenone 3, 6 and 12µg doses respectively as compared to vehicle (37.70±2.03) in SN (F=12.53, df=4, p=0.0001; n=4). THir level in SN (F=6.541, df=4, p=0.0030; n=4) in terms of Count/Area by rotenone 3, 6 and 12µg doses was 892.42±147.42, 636.11±121.05 and 455.10±57.36 respectively as compared to vehicle (1048.45±95.67). But significantly lowered THir only by 6 and 12µg doses both in terms of % Area and Count/Area in SN Fig 5(a) and 1(b). The decrement in THir indicates loss of neuronal cells.
Fig. 5 Graphical representation showing TH immunoreactivity in rat brain regions STR and SN [7 days after rotenone (3, 6 and 12μg) treatment] (a) %Area (b) Count/Area. Values are expressed as Means ± S.E.M and analyzed by ANOVA post hoc Newman-Keuls Multiple Comparison Test (n=4). **P<0.001 and * P <0.05 Vehicle (DMSO) vs Rotenone treated group.
GFAP immunofluorescence
GFAP immunofluorescence was performed to assess expression of GFAP, which is a phenotypic marker for astroglial cells. All the doses of rotenone 3, 6 and 12µg caused significantly enhanced GFAPir level in terms of % Area 2.95±0.73, 6.1±0.45 and 7.97±0.34 respectively as compared to vehicle (0.96±0.36) in SN (F=45.91, df=4, p<0.0001; n=4). The significant increase in GFAPir level in terms of Count/Area was 144.86±24.80, 196.57±11.90 and 248.0±4.53 respectively by rotenone 3, 6 and 12µg doses as compared to vehicle (93.52±14.60) in SN (F=21.53, df=4, p<0.0001; n=4). Rotenone 3, 6 and 12µg doses resulted in GFAPir level in terms of % Area 0.91±0.19, 1.34±0.28 and 1.55±0.40 respectively as compared to vehicle (0.42±0.07) in STR (F=4.885, df=4, p=0.0101; n=4), while GFAPir level in terms of Count/Area was 120.93±11.03, 132.77±14.03 and 151.76±19.88 respectively by rotenone 3, 6 and 12µg doses as compared to vehicle (87.05±2.81) in STR (F=7.632, df=4, p=0.0015; n=4). But only by higher doses 6 and 12µg of rotenone resulted significant increase in GFAPir level in STR Fig. 6 (a) and (b).
The increment in GFAPir indicates astroglial activation.
Fig. 6 Graphical representation showing GFAP immunoreactivity in rat brain regions STR and SN [7 days after rotenone (3, 6 and 12μg) treatment] (a) %Area (b) Count/Area. Values are expressed as Means ± S.E.M and analyzed by ANOVA post hoc Newman-Keuls Multiple Comparison Test (n=4). **P<0.001 and * P <0.05 Vehicle (DMSO) vs Rotenone treated group.
CD11b/c immunofluorescence
CD11b/c immunofluorescence was performed to assess expression of CD11b/c, which is a marker for microglial cells. All the doses of rotenone 3, 6 and 12µg produced significantly elevated, i.e. 4.52±1.09, 5.51±0.52 and 6.64±0.97 CD11b/c ir level in terms of % Area respectively as compared to vehicle (1.93±0.2) in SN (F= 9.110, df=4, p= 0.0006; n=4). The significantly increased CD11b/c ir level in terms of Count/Area was 455.18±52.34, 522.99±60.90 and 594.95±118.54 by all the doses of rotenone 3, 6 and 12µg respectively as compared to vehicle (212.24±14.52) in SN (F= 7.303, df=4, p= 0.0018; n=4). Rotenone 3, 6 and 12µg doses resulted in CD11b/c ir level
in terms of %Area 2.87±0.23, 3.30±0.70 and 3.75±1.086 respectively as compared to vehicle (2.26±0.43) in STR (F= 0.9988, df=4, p= 0.4386; n=4) while changes in terms of Count/Area was 237.49±62.97, 266.28±125.37 and 278.63±70.62 respectively as compared to vehicle (248.91±97.02) in STR (F= 0.1734, df=4, p=0.9486; n=4). But none of the changes were significant in STR Fig. 7 (a) and (b). The increased expression of CD11b/c ir indicates microglial activation.
Fig. 7 Graphical representation showing CD11b/c immunoreactivity in rat brain regions STR and SN [7 days after rotenone (3, 6 and 12μg) treatment] (a) %Area (b) Count/Area. Values are expressed as Means ± S.E.M and analyzed by ANOVA post hoc Newman-Keuls Multiple Comparison Test (n=4). **P<0.001 and * P <0.05 Vehicle (DMSO) vs Rotenone treated group.
Iba-1 immunofluorescence
Iba-1 immunofluorescence was performed to assess microglial cells in brain. Rotenone 3, 6 and 12µg doses caused Iba-1 ir level in terms of %Area 0.63±0.10, 0.78±0.18 and 0.74±0.24 respectively as compared to vehicle (0.62±0.25) in STR (F= 0.1535, df=4, p= 0.9585; n=4). The Iba-1 ir level in terms of Count/Area was 245.71±90.81, 223.083±94.51 and 255.08±134.52 by rotenone 3, 6 and 12µg doses respectively as compared to vehicle (270.17±97.02) in STR (F= 0.03970, df=4, p= 0.9967; n=4).While Iba-1 ir level in terms of %Area was 1.21±0.18, 2.25±0.27 and 3.82±0.65 by rotenone 3, 6 and 12µg doses respectively as compared to vehicle (0.82±0.18) in SN (F= 14.81, df=4, p<0.0001; n=4). The Iba-1 ir level in terms of Count/Area was 389.94±69.61, 607.74±120.20 and 729.76±122.84 by rotenone 3, 6 and 12µg doses respectively as compared to vehicle (257.97±58.51) in SN (F=
6.134, df=4, p= 0.0039; n=4). However, significantly elevated Iba-1 expression only by 6 and 12µg doses in SN both in terms of %Area as well as Count/Area Fig 8(a) and (b). The increased immunoreactivity of Iba-1 indicates microglial activation.
Fig. 8 Graphical representation showing Iba-1 immunoreactivity in rat brain regions STR and SN [7 days after rotenone (3, 6 and 12μg) treatment] (a) %Area (b) Count/Area. Values are expressed as Means ± S.E.M and analyzed by ANOVA post hoc Newman-Keuls Multiple Comparison Test (n=4). **P<0.001 and * P <0.05 Vehicle (DMSO) vs Rotenone treated group.
Caspase-3 mRNA expression
Apoptotic cell death was assessed by Caspase-3 gene expression, as it is the key executioner of apoptosis. Rotenone 3, 6 and 12µg significantly (F=12.28, df=4, p=0.0001; n=4) increased caspase-3 mRNA expression 1.56±0.16, 1.99±0.27 and 2.76±0.50 respectively in MB as compared to vehicle (0.57±0.08). While, in STR caspase-3 expression was 0.78±0.09, 1.23±0.12 and 2.06±0.23 as compared to vehicle (0.53±0.07), but significant difference (F=24.47, df=4, P<0.0001; n=4) was observed by 6 and 12µg doses only when compared to vehicle Fig. 9 (a) and (b). This is an indicative of involvement of apoptotic cell death differentially in the brain areas.
Fig. 9 mRNA expression of caspase-3 & β-actin [7 days after rotenone (3, 6 and 12μg) treatment] (a) Gel representative & 9(b) Graphical representation of caspase-3 expression w.r.t. to β-actin in rat brain regions, STR and MB. Data were expressed in mean ± S.E.M and analyzed by ANOVA post hoc Newman-Keuls Test (n=4). **P<0.001 and * P <0.05 Vehicle (DMSO) vs Rotenone treated group.
Discussion
Rotenone is known to induce neurotoxicity in rats by selectively degenerating dopaminergic neurons (Sherer et al.
2003b). Glial cells situated between vessels and neurons, acts as supporter for neurons from insults. Thus, glial cells are the targets and mediators of many insults to the nervous system. An important aspect of rotenone neurotoxicity i.e. whether the rotenone exhibits toxicity in neuronal and neuronal supportive cells in similar manner in different brain areas has been addressed in the present study. Therefore, an understanding of interaction between neuronal and neuronal supportive cells in different brain areas following rotenone was explored by using specific markers for neuronal and neuronal supportive cells. Rotenone was administered in the doses known to cause motor behavior impairment (Swarnkar et al. 2010, Swarnkar et al. 2011). The brain regions focused in this study are STR and MB, Selection of these brain regions based on involvement of these areas in the neurodegenerative diseases like PD, where dopaminergic neurons are mainly involved.
In normal conditions, neuronal cells have high content of tyrosine hydroxylase (TH), the rate-limiting enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to dihydroxy phenylalanine (DOPA) (Kaufman, 1995). DOPA is a precursor for dopamine that, in turn, is a precursor for another catecholamine (Norepinephrine). TH is found in the cytosol of all cells containing catecholamines and got depleted due to neuronal toxicity of dopaminergic neurons (Kaufman, 1995). In this study, neuronal changes were assessed with TH immunohistochemistry, and measured with TH protein levels. Based on the lowered TH level, the neurotoxicity was significantly apparent in MB while STR remains spared following rotenone treatment. The observation may be attributed to the local administration of rotenone in nigral region in MB, which is predominantly dopaminergic.
Even daily systemic intraperitoneal treatment of rotenone, over a period of 2 months was reported to reduce tyrosine hydroxylase immunoreactivity in striatum and caudate putamen (Alam and Schmidt, 2002).
We used GFAP-ir as a marker of reactive astrocytes for qualitative assessment, while GFAP protein level was measured to give a quantitative picture of astrogliosis. Astroglial activation occurred in STR and SN of all rotenone treated animals; even in the same brain region dopaminergic neurons are found unaffected. In these animals, activated astroglia were present throughout striatum and nigra and were not limited to the area of denervation. All the doses of rotenone caused increased level of GFAP in MB, while only the higher dose was effective in STR. GFAP-ir further confirmed the GFAP immunolabelling results, as in both STR and MB astroglial cell activation was observed. The morphological characteristic of astroglial cell activation- astrous and star shaped cells, were also detected in MB areas.
Rotenone exhibits reactive microgliosis and astrogliosis in rat spinal cord (Samantrey et al. 2007). It is relevant to mention that reactive microgliosis and astrogliosis have been demonstrated in patients of PD by MRI (Schwarz et al. 1996) and following administration of neurotoxins like 6-OHDA (He et al. 2001), LPS [Roy et al.
2006; Kim etb al. 2000) and MPTP (Wu et al. 2002; Du et al. 2001). Therefore, we assessed microglial activation, by using CD11b/c and Iba-1 protein markers. In the present study rotenone resulted in microglial cell activation as visualized with CD11b/c and Iba-1 immunohistochemistry, and measured with CD11b/c and Iba-1 protein levels.
Rotenone infusion caused microglial activation but only in MB area, while in STR no significant changes were observed. Image-1 showed the result of IF in STR indicating increased GFAP-ir by higher doses of rotenone, while none of the dose of rotenone caused apparent change in TH, CD11b/C and Iba-1 immunoreactivity. Image-2 demonstrated the result of IF in SN indicating decreased TH-ir, and increased GFAP, CD11b/C and Iba-1 immunoreactivity by all the doses of rotenone as compared to vehicle treated group. The IF results correlated with the observations from western blotting experiments. It is observed that rotenone results in differential expression of specific marker proteins in both brain areas with variability in terms of dose.
Swarnkar et al. (2011) previously reported SN is more vulnerable to damage by rotenone and possible reason might be higher susceptibility of SN for free radicals. Neuronal loss in response to rotenone infusion was pronounced in SN. In both the regions activation of microglial cells was less pronounced as compared to that of astroglial cells. These results suggest that reactive astrocytosis plays more dominant role than microgliosis caused by rotenone toxicity. The differential effect of rotenone may also be attributed to the fact that neurons depend more on mitochondrial respiration in comparison to astrocytes that can effectively upregulate glycolysis in order to maintain normal ATP levels. Furthermore, astrocytes are known to upregulate de novo synthesis of glutathione on demand (Węgrzynowicz et al. 2007).
Caspase-3 gene expression was done to assess the apoptotic cell death in rat brain areas. The caspase-3 expression was more pronounced with higher doses of rotenone in both STR and MB areas, while lowest dose resulted in increased caspase-3 expression only in MB. Caspase-3 expression was regulated in response to dopaminergic neuronal death (Dodel et al. 1999). The pattern of caspase-3 gene expression found correlated with astroglial cell activation as observed in terms of dose dependence and brain area affected. Thus Caspase-3 elevation by rotenone induces apoptosis and astroglial cell activation appears to be interlinked.
The present study indicates that rotenone toxicity appears first in neuronal supportive cells rather in neuronal cells. Both the brain areas responded to rotenone differentially in terms of dose and cell involvement. We demonstrate here a preferred specificity of rotenone action towards neurons in MB than STR. It suggests that neuronal supportive cells are more affected in comparison to neuronal cells. Since astroglial activation occurred in both the brain regions of all rotenone-treated animals, astroglia may play a primary role in rotenone-induced neurodegeneration. Astroglial activation, through ROS production, may contribute to selective oxidative damage that may underlie rotenone toxicity as observed in our study (Swarnkar et al. 2012b).
Conclusion
In conclusion, astroglial activation and apoptosis appeared as a major feature in rotenone-induced neurotoxicity.
Based on the changes in the markers for both neuronal and glial cells, MB appeared as more sensitive than STR towards rotenone induced cell toxicity because there was significant effect on TH (neuronal marker), CD11b/c and Iba-1 (Microglial markers), and GFAP (astroglial marker) in MB while only GFAP was increased in STR. This also indicates that astroglial cells are more prone to rotenone toxicity than neuronal and microglial cells. The study further supports the concept that majority of neuronal supportive cells in the CNS are no longer passive partners to neurons, which has opened a new approach to understand neurodegenerative disorders.
Acknowledgments: Authors are thankful to Dr. V. P. Nakka and Mr. S.M. Verma for their help in histological studies and supporting this work. Author SS gratefully acknowledges the Council of Scientific and Industrial Research (CSIR), India for research fellowship. CDRI Communication No. 8351
Conflict of interest: The authors declare that they have no conflict of interest.
Declaration: The authors do not have financial relationship with the organization for the research.
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Table-1: Primer sequences and specific conditions for RT-PCR study of Caspase-3 and β-actin Figure Legends:
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