Protective role of curcumin against sulfiteinduced structural changes in rats’ medial prefrontal cortex Ali Noorafshan 1,2, Reza Asadi-Golshan 2, Mohammad-Amin Abdollahifar 2, Saied Karbalay-Doust 1,2 1
Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, Anatomy Department, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
2
Objectives: Sodium metabisulfite as a food preservative can affect the central nervous system. Curcumin, the main ingredient of turmeric has neuroprotective activity. This study was designed to evaluate the effects of sulfite and curcumin on the medial prefrontal cortex (mPFC) using stereological methods. Methods: Thirty rats were randomly divided into five groups. The rats in groups I–V received distilled water, olive oil, curcumin (100 mg/kg/day), sodium metabisulfite (25 mg/kg/day), and sulfite + curcumin, respectively, for 8 weeks. The brains were subjected to the stereological methods. Cavalieri and optical disector techniques were used to estimate the total volume of mPFC and the number of neurons and glial cells. Intersections counting were applied on the thick vertical uniform random sections to estimate the dendrites length, and classify the spines. Non-parametric tests were used to analyze the data. Results: The mean mPFC volume, neurons number, glia number, dendritic length, and total spines per neuron were 3.7 mm3, 365 000, 180 000, 1820 μm, and 1700 in distilled water group, respectively. A reduction was observed in the volume of mPFC (∼8%), number of neurons (∼15%), and number of glia (∼14%) in mPFC of the sulfite group compared to the control groups (P < 0.005). Beside, dendritic length per neuron (∼10%) and the total spines per neuron (mainly mushroom spines) (∼25%) were reduced in the sulfite group (P < 0.005). Discussion: The sulfite-induced structural changes in mPFC and curcumin had a protective role against the changes in the rats. Keywords: Curcumin, Sulfite, Cortex, Stereology, Rat
Introduction Sulfite salts are broadly used as food additives and have been believed to be safe by Food and Drug Administration since 1959. Sodium metabisulfite (Na2S2O5), potassium metabisulfite, sodium bisulfite, potassium sulfite, and sodium sulfite are five kinds of sulfite salts that are widely used as preservatives in food and drug preparations. Previous studies have reported that the ingested sulfite is absorbed by the gastrointestinal tube and distributed essentially to all body organs including the brain.1 Ingested sulfite salts generate bisulfite (HSO−3), sulfite (SO3−2), and sulfur dioxide (SO2) by reacting with water. Considerable endogenous amount of sulfites is generated in the body by metabolism of the amino acids that contain sulfurs, such as methionine and cysteine. Correspondence to: Saied Karbalay-Doust, Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Zand Avenue, Shiraz 71348-45794, Iran. Email: [email protected]
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© W. S. Maney & Son Ltd 2015 DOI 10.1179/1476830514Y.0000000123
Therefore, humans are exposed to sulfites both endogenously and exogenously. Both endogenous and exogenous sulfites can cause toxicity via reacting with a lot of humoral and cellular components.2–4 Therefore, it can be suggested that neurons are susceptible to toxicity of sulfites. It has also been reported that liver, kidney, and heart have high sulfite oxidase activities, whereas spleen, testes, and brain tissues have shown very low activities.5 Up to now, studies have been limited to special brain regions, including hippocampus structure and function, and medial prefrontal cortex (mPFC) has received little attention.3,6–8 Curcumin, the main element of turmeric, is known to have multiple properties, including anti-oxidant, anti-inflammatory, anti-tumor, and anti-cancer effects. In addition, it has been suggested to be of use in treatment of several diseases, such as central nervous system disorders.9 At least 10 known
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neuroprotective actions of curcumin were reported by previous studies and many of these actions might be realized in vivo.10 Indeed, previous studies showed that dietary curcumin was a strict candidate to be used in the prevention or treatment of major disabling age-related neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and stroke.11 Researchers annually published about 200 papers concerning curcumin in the last decade; of course, they were only related to pharmaceutical preparations. A critical scrutiny of the recent wave of scientific reports on curcumin clearly suggests that curcumin acts as a potent modulator of the activity of many vital biomacromolecular targets involved in homeostasis of mammalian physiology.12 Curcumin was considered to be examined in this study because of being the main part of turmeric and that it can be easily added to foods.8 The mPFC plays a role in cognitive function, memory, and learning. This study was conducted to evaluate the effects of sulfite and curcumin on the structure of mPFC, including its total volume, number of neurons and glial cells, dendritic length, and spines in mPFC using modern stereological techniques.
Materials and methods Animals This study was carried out on 30 healthy male Sprague–Dawley rats weighing 250–280 g. All the trial procedures conducted on the animals were performed in accordance with the standards established by the Animal Care and Ethics Committee at Medical School, Shiraz University of Medical Sciences, Shiraz, Iran (Agreement License No. 905954). The animals were randomly categorized into five experimental groups each containing six rats. The rats in groups I–V received daily gastric gavages of distilled water, olive oil, curcumin (100 mg/kg/ day), sodium metabisulfite (25 mg/kg/day), and sulfite + curcumin, respectively, for 8 weeks. Distilled water and olive oil were the solvents of sodium metabisulfite and curcumin, respectively. The dose of sodium metabisulfite used in this study was selected according to the acceptable daily intake which is 163 mg/day.3,13 Besides, the dose of curcumin was designated according to the appropriate dose of curcumin with no side effects on the liver, kidney, and blood levels of aspartate aminotransferase, alanine aminotransferase, urea nitrogen, and creatinine.14
Tissue preparation At the end of the experiment, the brains were quickly removed from the skull. Then, the right hemispheres were immersed in neutral buffered formalin. After tissue processing, embedding in paraffin, and coronal serial sectioning of 26 μm thick, they were stained
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using cresyl violet (1% cresyl violet acetate in distilled water) and were used to estimate the number of neurons and glial cells as well as the volume of the mPFC. Besides, the slabs of the left hemispheres were processed for Golgi impregnation procedure.15
Estimation of the mPFC volume Medial PFC estimates were accomplished on the sections at the first appearance of the most anterior section where the underlying white matter appeared (Bregma 4.20 mm) and were followed until the appearance of the decussating of the corpus callosum (Bregma 2.28 mm) according to the atlas of Paxinos and Watson.16 After all, 8–12 sections per mPFC were analyzed at the final magnification of 25× (Fig. 1). The mPFC can be subdivided into infralimbic (IL), prelimbic (PL), and dorsal anterior cingulate cortex (AC) (Fig. 1).17 However, all the three regions were examined as a whole because of the difficulty to reliably define the boundaries of these regions. The area of interest included all the cortical layers (two, three, five, and six). Nevertheless, layer 1 was not included because of containing very few cells.18 It should be mentioned that mPFC does not have a fourth layer. The total volume of the mPFC was estimated using Cavalieri’s principle (Fig. 1).19–21 The software designed at the Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences was used for estimating the parameters. In this way, the product of the areas and the measured tissue thickness (T) between the saved sections were calculated. The area was estimated using point-counting method. The area per point (a/ p) was 0.04 mm2 and averagely 450–500 points were counted per animal. The following formula was used for estimating the volume: V (mPFC) =
a × P(mPFC) × T. p
Estimation of the number of neurons and glial cells As described previously, the optical disector was used for estimating the total number of neurons and glial cells in the mPFC (Fig. 1).19–21 The area of mPFC was demarcated and counts were made at random locations. The area of the counting frame (a/f ) was 35 × 35 μm2. Section thickness was used as the height of the disector (h), excluding the 4 μm thick guard zones at the top and bottom of each section using a microcator (MT 12, Heidenhain, Germany). To calculate the suitable guard zone, Z-axis distribution of the nuclei was plotted. The counted neurons were scored and categorized into 10 histograms from the 0–100 percentiles through the brain tissue section from the upper (0%) to the lower surface (100%). Fig. 1 shows
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Figure 1 Estimation of the volume and number of mPFC. (A) An illustration of sectioning used to estimate the volume of the mPFC. The borders of the mPFC and subareas (AC: anterior cingulate; PL: prelimbic; IL: infralimbic). (B) Point-counting methods. (C) Z-axis distribution of the neuronal nuclei. The counted neurons were scored and categorized into 10 histograms from the percentiles 0–100 through the tissue section. The vertical dashed line indicates how the particles would present if all the visible particles were equally distributed throughout the z-axis. (D) An unbiased counting frame was superimposed on the images of the mPFC. (E) Any cells whose nuclei appeared during scanning of the height of the disector and lied on the frame or its accepted borders (dotted line) was counted (arrow).
the z-axis distribution of the nuclei. The upper and lower 20% of the histogram were considered as the guard zones and the counting box was placed on the remaining 60% (h). According to the histogram, under-sampling was well adjusted and corrected. Any nucleus coming into the maximal focus within the next focal sampling plane was selected if it was located completely or partly inside the counting frame and did not touch the exclusion line (Fig. 1). Post-shrinkage thickness was measured during cell counting and was used to determine an average thickness of 20 μm (t). Both neurons and glial cells were distinguished based on the differences in their morphological characteristics and were separately counted within the frame.22 In order to determine the cell densities, neurons, or glial cells counted in the mPFC were divided by the total volume of the counting frames
Nv (cells/mPFC) =
t Q− × , a BA ×h P× f
where ‘ΣQ −’ was the number of the nuclei coming into focus during scanning the ‘h’, ‘ΣP’ was the total count of the unbiased counting frame in all fields, ‘h’ was the 250
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height of the disector, ‘a/f’ was the frame area, ‘t’ was the mean section thickness, and ‘BA’ was the block advance of the microtome which was set at 26 μm. On the average, 340–550 neurons and 130–260 glial cells were counted per mPFC. The total number of the neurons was estimated by multiplying the numerical density (Nv) by the V (mPFC).
Estimation of the coefficient of error (CE) The CE for the estimate of the volume is the function of the noise effect and systematic random sampling variance for the sums of the estimated areas. In this study, the cross-sectional areas of the mPFC were estimated by point counting, and CE(V ) was calculated using the following formula:23 −1 CE(V ) = P 1 2 3 Pi + × Pi Pi+2 − 4 Pi Pi+1 240 b +0.0724 × √ × a
1
2 n Pi ,
where ‘b’ and ‘a’ represented the mean section boundary length and mean sectional area, respectively. The
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Table 1 Coefficients of error (CE) for total volume, neuronal, and glial numbers as well as neuronal and glial densities in the mPFC
Groups Distilled water Olive oil Curcumin Sulfite Sulfite + curcumin
Neuron Density
Number
Density
Number
0.03 0.03 0.03 0.04 0.04
0.04 0.03 0.03 0.04 0.04
0.05 0.05 0.05 0.06 0.05
0.04 0.04 0.04 0.05 0.05
0.05 0.06 0.05 0.06 0.06
CE for the estimate of the total neuron number, CE(N), was calculated using CE(V) and CE(Nv) as follows: 1
CE(N) ) + CE (V ) 2, = CE (Nv − 2 n Q × CE(Nv) = 2 n−1 Q−
2
Glia
mPFC volume
2
1 2 2 Q− P (P )2 . + 2 − − Q P P The CEs have been shown in Table 1.
Estimation of the dendritic length Vertical uniform random sectioning is necessary for length estimation and it was achieved as follows.19–21,25,26 Briefly, 9–10 cylinders were punched out using a trocar with 1 mm diameter vertical to the pial surface of the cortex (Fig. 2). The cylinders were randomly rotated along their vertical axes and embedded in one paraffin block. Then, 100 μm thickness slabs were taken using a microtome. The mean dendritic length per neuron was calculated using the following formula: I N = Total dendritic length in the population . Total number of neurons in the population A fixed slab height of 100 μm was scanned using a microscope (Nikon E-200, Japan) equipped with an objective lens of 100× and numerical aperture of 1.4 connected to a computer. A cycloid grid and a counting frame were superimposed on the monitor image (Fig. 2). Overall, two quantities were measured: (i) number (Q −) of the cell bodies of the neurons using the optical disector method, and (ii) the total number of the intersections (I) between the dendrites axes and the cycloid oriented parallel to the vertical axis of the cylinder (Fig. 2). The following formula was used: I I N = 2 × a 1 M −1 , Q l asf −
a where ‘ ’ was the test area per cycloid test length, ‘asf’ l was the area associated with cycloid grid divided by the area of the counting frame, and ‘M’ was the final magnification at ×4000.26
Estimation of density and morphology of the dendritic spines To estimate the density and morphology of the spines, the above-mentioned dendrites were considered. Dendritic spines were identified as small protrusion that drawn out of the parent dendrite for less than 3 μm. Dendritic protrusion was classified as spines when they exhibited enlargement at the tip and presented stubby or mushroom-type forms. On the other hand, the spines without enlargement were defined as thin filopodia-like protrusions. The spines were counted only if they looked continuous with the parent dendrite. Density and morphology of the spines were quantified and stated as the number of spines per neuron.27
Statistical analysis The data were analyzed using non-parametric tests, including Kruskall–Wallis and Mann–Whitney U-test. Besides, P < 0.05 was considered as statistically significant.
Results Volume of the right mPFC A significant ∼8% reduction was observed in the total volume of mPFC in the sulfite-treated rats compared to the control ones (P < 0.005; Fig. 3). These findings demonstrated that concomitant treatment of curcumin during sulfite consumption prevented the mPFC volume loss induced by sulfite.
Neuron and glial cell counts (hemispheric) A significant difference was found among the study groups regarding the total number of neurons as well as the total number of glial cells (P < 0.005). Moreover, analysis of the mean neuron number revealed a significant decrease (∼15%) in the sulfite-treated group compared to the control groups (P < 0.005; Fig. 3). Likewise, the mean glial cell number was significantly lower (∼14%) in the sulfite group compared to the control groups (P < 0.005; Nutritional Neuroscience
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Figure 2 Estimation of the dendrites’ length. (A) The vertical cylinders were punched out from the MPF cortex perpendicular to its pial surface. (B) The cylinder was randomly rotated along its vertical axis, sectioned using a microtome, and mounted on a slide. (C) Four cycloids were placed at a rectangle. The length of each cycloid was identical to twice the length of the minor axis (r). The area associated with the cycloids was calculated by multiplying the X by Y. (D, E) When the sections were scanned, the number of the cell bodies of the neurons was counted using the optical disector method and unbiased counting frame. The total number of the intersections between the dendrites axes and the cycloid were counted (arrow head). The cycloid was oriented parallel to the vertical axis (arrow).
Fig. 3). The study results revealed that curcumin prevented neuron and glial cell loss induced by sodium metabisulfite.
Dendritic length measures On the average, a 10% decrease was observed in the length of dendrites per neuron in the rats treated with sulfite in comparison to those receiving distilled water, olive oil, or curcumin (P < 0.005). In addition, the concomitant treatment of sulfite + curcumin prevented the loss of dendritic length in rats (P < 0.005; Fig. 3).
Density and morphology of the spines The results showed that the total spine density was averagely reduced by ∼25% in the sulfite group in comparison to the control groups (P < 0.005; Fig. 4). Besides, the density of the mushroom spines was averagely reduced by ∼45% in the sulfite-treated rats in comparison to the control ones (P < 0.005; Fig. 4). Nonetheless, the stubby and thin spines of the mPFC were remained unchanged (Fig. 4). The concomitant treatment of sulfite + curcumin prevented the loss of dendritic spines in the mPFC.
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Discussion The present study evaluated the effects of treatment with sodium metabisulfite at the first step and showed a significant reduction in the volume of mPFC, a significant loss of neurons and glial cells, and reduction of dendritic length and spines in mPFC. Our earlier observations also showed that sodium metabisulfite could induce learning and memory changes in rats.8 Medial PFC has a key role in these tasks. Therefore, physiological changes are accompanied by histological alterations. Of course, neuron loss or physiological alterations are not limited to mPFC. One study reported that sulfite could cause the loss of hippocampal neurons in rats.3 Toxic effects of sulfite, both in vivo and in vitro, have been reported by various studies. For instance, a synergistic toxic effect on mesencephalic cell lines has been shown by sulfite and peroxynitrite anion.3 Moreover, sulfite treatment was revealed to cause impairment of the avoidance performance and increase the excitability of the spinal reflexes.6 Another study also reported that sodium metabisulfite caused neurotoxicity due to the increased lipid peroxidation in the brain tissue.3 A good justification for this neuronal
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Figure 3 The dot plot presents the total volume of the mPFC (A), total number of the neurons (B) and the glial cells (C), and dendritic length per neuron (D) in the mPFC in the rats receiving distilled water, olive oil, curcumin, and sulfite with or without curcumin treatment. Each dot represents the parameters in an animal.
toxicity of sulfite is that the brain tissue has low sulfite oxidase activity compared to other tissues. Another explanation is that extreme neurological abnormalities were observed in case of sulfite oxidase deficiency. Sulfite oxidase plays a protective role in detoxifying sulfite and low levels of expression of this enzyme may predispose neurons to damage by excess sulfite.5 There is little information about the mechanism of sulfite toxicity in the nervous tissue, but its toxic effect on the central nervous system may involve formation of sulfur and oxygen-centered free radicals.13 Previous reports suggested that various peroxidases could oxidize sulfite and also sulfite could be oxidized non-enzymatically. Non-enzymatic oxidation or oxidation by peroxidase of sulfite occurs via one electron oxidation. In this case, a sulfur trioxide radical is produced from sulfite during sulfite metabolism. It has been reported that sulfur trioxide radical may be involved in many toxic effects of sulfite, including destruction of amino acids, impairment of DNA synthesis, and increase of lipid peroxidation.28 In addition, it can be proposed that cysteine-S-sulfate, a brain damaging metabolite in sulfite oxidase deficiency, could be responsible for the observed toxic effects of sulfite. It should be mentioned that
cysteine-S-sulfate does not normally exist in human body.29 At the second step of the study, the protective effect of curcumin was evaluated. The findings showed that concomitant treatment of curcumin during sulfite consumption prevented the reduction in the volume of mPFC, loss of neurons and glial cells, and reduction of dendritic length and spines in mPFC. This finding is in line with our earlier observations showing that curcumin prevented learning and memory impairment induced by sulfite in rats.8 This finding is also in accordance with the previous studies reporting the neuroprotective effects of curcumin. Overall, there is a vast range of protective functions of curcumin in nervous system injuries, including improvement in lead-induced memory changes in rats, improvement in dorsal root ganglion and sciatic nerve structure and function after crush, improvement in behavioral changes after chronic stress in rats, and prevention of neuropathological changes in the hippocampus in an animal model of Alzheimer’s disease.14,30,31 Studies have also shown that different doses of curcumin decreased the oxidized proteins and interleukin1beta which is a proinflammatory cytokine increasing in the brains of these mice. Furthermore, treatment with curcumin was reported to improve the
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Figure 4 Density of the dendritic spines. The density of thin (A), mushroom (B), and stubby (C) spine per neuron and total spine density (D) in the mPFC of different groups are shown. The significant difference between the sulfite-treated group and other groups is indicated. *P < 0.005. DW: distilled water; OO: olive oil; CUR: curcumin; SUL: sulfite; SUL + CUR: sulfite + curcumin.
colchicines-induced cognitive impairment.14 Also, it could significantly reduce the colchicines-induced elevated lipid peroxidation. In summary, extensive studies have shown the protective effect of curcumin in almost all the bodily disorders due to its many useful properties, including anti-inflammatory, antihypertensive, anti-hyperlipidemic, anti-tumor, anticancer, anti-phlogistic, anti-diabetic, anti-psoriasis, anti-thrombotic, anti-hepatotoxic, and anti-microbial effects.9–11,14 Thus, the neuroprotective effects of curcumin against sulfite observed in our study might have resulted from its anti-oxidant, anti-inflammatory, anti-apoptotic, and anti-ischemic properties.
Conclusion Curcumin could prevent loss of neurons, glial cells, reduction of the total volume of the rats’ mPFC, and reduction of dendritic length and spines induced by sulfite ingestion. This finding agrees with the previous reports showing the neurotoxic effect of sulfite and neuroprotective effect of curcumin.
Acknowledgements This work was financially supported by grant no. 905954 from Shiraz University of Medical Sciences, Shiraz, Iran. The work was performed at
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Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. This article was a part of the thesis written by Reza Asadi-Golshan, MSc student of Anatomy. Hereby, the authors would like to thank Ms A. Keivanshekouh at the Research Improvement Center of Shiraz University of Medical Sciences for improving the use of English in the manuscript. Source of financial support: This work was financially supported by grant No. 90-5954 from Shiraz University of Medical Sciences, Shiraz, Iran.
Disclaimer statements Contributors Designer and idea: Ali Noorafshan; Executive: Saied Karbalay-Doust; Animal experiment and stereological estimations: Reza Asadi-Golshan, Mohammad-Amin Abdollahifar. Funding None. Conflict-of-Interest None. Ethics Approval All the trial procedures conducted on the animals were performed in accordance with the standards established
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by the Animal Care and Ethics Committee at Medical School, Shiraz University of Medical Sciences, Shiraz, Iran (Agreement License No. 90-5954).
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17 Czéh B, Perez-Cruz C, Fuchs E, Flügge G. Chronic stressinduced cellular changes in the medial prefrontal cortex and their potential clinical implications: does hemisphere location matter? Behav Brain Res 2008;190:1–13. 18 Koss WA, Sadowski RN, Sherrill LK, Gulley JM, Juraska JM. Effects of ethanol during adolescence on the number of neurons and glia in the medial prefrontal cortex and basolateral amygdala of adult male and female rats. Brain Res 2012;1466: 24–32. 19 Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Møller A, Nielsen K, et al. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 1988;96:379–94. 20 Gundersen HJ, Bagger P, Bendtsen TF, Evans SM, Korbo L, Marcussen N, et al. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 1988;96: 857–81. 21 Kristiansen SL, Nyengaard JR. Digital stereology in neuropathology. APMIS 2012;120:327–40. 22 Rubinow MJ, Juraska JM. Neuron and glia numbers in the basolateral nucleus of the amygdala from preweaning through old age in male and female rats: a stereological study. J Comp Neurol 2009;512:717–25. 23 Gundersen HJ, Jensen EB. The efficiency of systematic sampling in stereology and its prediction. J Microsc 1987;147: 229–63. 24 Braendgaard H, Evans SM, Howard CV, Gundersen HJ. The total number of neurons in the human neocortex unbiasedly estimated using optical disectors. J Microsc 1990; 157:285–304 25 Baddeley AJ, Gundersen HJ, Cruz-Orive LM. Estimation of surface area from vertical sections. J Microsc 1986;142:259–76. 26 Howard CV, Cruz-Orive LM, Yaegashi H. Estimating neuron dendritic length in 3D from total vertical projections and from vertical slices. Acta Neurol Scand Suppl 1992;137:14–9. 27 Chapleau CA, Carlo ME, Larimore JL, Pozzo-Miller L. The actions of BDNF on dendritic spine density and morphology in organotypic slice cultures depend on the presence of serum in culture media. J Neurosci Methods 2008;169:182–90. 28 Ribera D, Jonker D, Narbonne JF, O’Brien J, Antignac E. Absence of adverse effects of sodium metabisulphite in manufactured biscuits: results of subacute (28-days) and subchronic (85-days) feeding studies in rats. Food Addit Contam 2001;18: 103–14. 29 Olney JW, Misra CH, de Gubareff T. Cysteine-S-sulfate: brain damaging metabolite in sulfite oxidase deficiency. J Neuropathol Exp Neurol 1975;34:167–77. 30 Pan R, Qiu S, Lu DX, Dong J. Curcumin improves learning and memory ability and its neuroprotective mechanism in mice. Chin Med J (Engl) 2008;121:832–39. 31 Noorafshan A, Abdollahifar MA, Karbalay-Doust S, AsadiGolshan R, Rashidiani-Rashidabadi A. protective effects of curcumin and sertraline on the behavioral changes in chronic variable stress-induced rats. Exp Neurobiol 2013;22:96–106.
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Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, Anatomy Department, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
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Objectives: Sodium metabisulfite as a food preservative can affect the central nervous system. Curcumin, the main ingredient of turmeric has neuroprotective activity. This study was designed to evaluate the effects of sulfite and curcumin on the medial prefrontal cortex (mPFC) using stereological methods. Methods: Thirty rats were randomly divided into five groups. The rats in groups I–V received distilled water, olive oil, curcumin (100 mg/kg/day), sodium metabisulfite (25 mg/kg/day), and sulfite + curcumin, respectively, for 8 weeks. The brains were subjected to the stereological methods. Cavalieri and optical disector techniques were used to estimate the total volume of mPFC and the number of neurons and glial cells. Intersections counting were applied on the thick vertical uniform random sections to estimate the dendrites length, and classify the spines. Non-parametric tests were used to analyze the data. Results: The mean mPFC volume, neurons number, glia number, dendritic length, and total spines per neuron were 3.7 mm3, 365 000, 180 000, 1820 μm, and 1700 in distilled water group, respectively. A reduction was observed in the volume of mPFC (∼8%), number of neurons (∼15%), and number of glia (∼14%) in mPFC of the sulfite group compared to the control groups (P < 0.005). Beside, dendritic length per neuron (∼10%) and the total spines per neuron (mainly mushroom spines) (∼25%) were reduced in the sulfite group (P < 0.005). Discussion: The sulfite-induced structural changes in mPFC and curcumin had a protective role against the changes in the rats. Keywords: Curcumin, Sulfite, Cortex, Stereology, Rat
Introduction Sulfite salts are broadly used as food additives and have been believed to be safe by Food and Drug Administration since 1959. Sodium metabisulfite (Na2S2O5), potassium metabisulfite, sodium bisulfite, potassium sulfite, and sodium sulfite are five kinds of sulfite salts that are widely used as preservatives in food and drug preparations. Previous studies have reported that the ingested sulfite is absorbed by the gastrointestinal tube and distributed essentially to all body organs including the brain.1 Ingested sulfite salts generate bisulfite (HSO−3), sulfite (SO3−2), and sulfur dioxide (SO2) by reacting with water. Considerable endogenous amount of sulfites is generated in the body by metabolism of the amino acids that contain sulfurs, such as methionine and cysteine. Correspondence to: Saied Karbalay-Doust, Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Zand Avenue, Shiraz 71348-45794, Iran. Email: [email protected]
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Therefore, humans are exposed to sulfites both endogenously and exogenously. Both endogenous and exogenous sulfites can cause toxicity via reacting with a lot of humoral and cellular components.2–4 Therefore, it can be suggested that neurons are susceptible to toxicity of sulfites. It has also been reported that liver, kidney, and heart have high sulfite oxidase activities, whereas spleen, testes, and brain tissues have shown very low activities.5 Up to now, studies have been limited to special brain regions, including hippocampus structure and function, and medial prefrontal cortex (mPFC) has received little attention.3,6–8 Curcumin, the main element of turmeric, is known to have multiple properties, including anti-oxidant, anti-inflammatory, anti-tumor, and anti-cancer effects. In addition, it has been suggested to be of use in treatment of several diseases, such as central nervous system disorders.9 At least 10 known
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neuroprotective actions of curcumin were reported by previous studies and many of these actions might be realized in vivo.10 Indeed, previous studies showed that dietary curcumin was a strict candidate to be used in the prevention or treatment of major disabling age-related neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and stroke.11 Researchers annually published about 200 papers concerning curcumin in the last decade; of course, they were only related to pharmaceutical preparations. A critical scrutiny of the recent wave of scientific reports on curcumin clearly suggests that curcumin acts as a potent modulator of the activity of many vital biomacromolecular targets involved in homeostasis of mammalian physiology.12 Curcumin was considered to be examined in this study because of being the main part of turmeric and that it can be easily added to foods.8 The mPFC plays a role in cognitive function, memory, and learning. This study was conducted to evaluate the effects of sulfite and curcumin on the structure of mPFC, including its total volume, number of neurons and glial cells, dendritic length, and spines in mPFC using modern stereological techniques.
Materials and methods Animals This study was carried out on 30 healthy male Sprague–Dawley rats weighing 250–280 g. All the trial procedures conducted on the animals were performed in accordance with the standards established by the Animal Care and Ethics Committee at Medical School, Shiraz University of Medical Sciences, Shiraz, Iran (Agreement License No. 905954). The animals were randomly categorized into five experimental groups each containing six rats. The rats in groups I–V received daily gastric gavages of distilled water, olive oil, curcumin (100 mg/kg/ day), sodium metabisulfite (25 mg/kg/day), and sulfite + curcumin, respectively, for 8 weeks. Distilled water and olive oil were the solvents of sodium metabisulfite and curcumin, respectively. The dose of sodium metabisulfite used in this study was selected according to the acceptable daily intake which is 163 mg/day.3,13 Besides, the dose of curcumin was designated according to the appropriate dose of curcumin with no side effects on the liver, kidney, and blood levels of aspartate aminotransferase, alanine aminotransferase, urea nitrogen, and creatinine.14
Tissue preparation At the end of the experiment, the brains were quickly removed from the skull. Then, the right hemispheres were immersed in neutral buffered formalin. After tissue processing, embedding in paraffin, and coronal serial sectioning of 26 μm thick, they were stained
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using cresyl violet (1% cresyl violet acetate in distilled water) and were used to estimate the number of neurons and glial cells as well as the volume of the mPFC. Besides, the slabs of the left hemispheres were processed for Golgi impregnation procedure.15
Estimation of the mPFC volume Medial PFC estimates were accomplished on the sections at the first appearance of the most anterior section where the underlying white matter appeared (Bregma 4.20 mm) and were followed until the appearance of the decussating of the corpus callosum (Bregma 2.28 mm) according to the atlas of Paxinos and Watson.16 After all, 8–12 sections per mPFC were analyzed at the final magnification of 25× (Fig. 1). The mPFC can be subdivided into infralimbic (IL), prelimbic (PL), and dorsal anterior cingulate cortex (AC) (Fig. 1).17 However, all the three regions were examined as a whole because of the difficulty to reliably define the boundaries of these regions. The area of interest included all the cortical layers (two, three, five, and six). Nevertheless, layer 1 was not included because of containing very few cells.18 It should be mentioned that mPFC does not have a fourth layer. The total volume of the mPFC was estimated using Cavalieri’s principle (Fig. 1).19–21 The software designed at the Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences was used for estimating the parameters. In this way, the product of the areas and the measured tissue thickness (T) between the saved sections were calculated. The area was estimated using point-counting method. The area per point (a/ p) was 0.04 mm2 and averagely 450–500 points were counted per animal. The following formula was used for estimating the volume: V (mPFC) =
a × P(mPFC) × T. p
Estimation of the number of neurons and glial cells As described previously, the optical disector was used for estimating the total number of neurons and glial cells in the mPFC (Fig. 1).19–21 The area of mPFC was demarcated and counts were made at random locations. The area of the counting frame (a/f ) was 35 × 35 μm2. Section thickness was used as the height of the disector (h), excluding the 4 μm thick guard zones at the top and bottom of each section using a microcator (MT 12, Heidenhain, Germany). To calculate the suitable guard zone, Z-axis distribution of the nuclei was plotted. The counted neurons were scored and categorized into 10 histograms from the 0–100 percentiles through the brain tissue section from the upper (0%) to the lower surface (100%). Fig. 1 shows
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Figure 1 Estimation of the volume and number of mPFC. (A) An illustration of sectioning used to estimate the volume of the mPFC. The borders of the mPFC and subareas (AC: anterior cingulate; PL: prelimbic; IL: infralimbic). (B) Point-counting methods. (C) Z-axis distribution of the neuronal nuclei. The counted neurons were scored and categorized into 10 histograms from the percentiles 0–100 through the tissue section. The vertical dashed line indicates how the particles would present if all the visible particles were equally distributed throughout the z-axis. (D) An unbiased counting frame was superimposed on the images of the mPFC. (E) Any cells whose nuclei appeared during scanning of the height of the disector and lied on the frame or its accepted borders (dotted line) was counted (arrow).
the z-axis distribution of the nuclei. The upper and lower 20% of the histogram were considered as the guard zones and the counting box was placed on the remaining 60% (h). According to the histogram, under-sampling was well adjusted and corrected. Any nucleus coming into the maximal focus within the next focal sampling plane was selected if it was located completely or partly inside the counting frame and did not touch the exclusion line (Fig. 1). Post-shrinkage thickness was measured during cell counting and was used to determine an average thickness of 20 μm (t). Both neurons and glial cells were distinguished based on the differences in their morphological characteristics and were separately counted within the frame.22 In order to determine the cell densities, neurons, or glial cells counted in the mPFC were divided by the total volume of the counting frames
Nv (cells/mPFC) =
t Q− × , a BA ×h P× f
where ‘ΣQ −’ was the number of the nuclei coming into focus during scanning the ‘h’, ‘ΣP’ was the total count of the unbiased counting frame in all fields, ‘h’ was the 250
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height of the disector, ‘a/f’ was the frame area, ‘t’ was the mean section thickness, and ‘BA’ was the block advance of the microtome which was set at 26 μm. On the average, 340–550 neurons and 130–260 glial cells were counted per mPFC. The total number of the neurons was estimated by multiplying the numerical density (Nv) by the V (mPFC).
Estimation of the coefficient of error (CE) The CE for the estimate of the volume is the function of the noise effect and systematic random sampling variance for the sums of the estimated areas. In this study, the cross-sectional areas of the mPFC were estimated by point counting, and CE(V ) was calculated using the following formula:23 −1 CE(V ) = P 1 2 3 Pi + × Pi Pi+2 − 4 Pi Pi+1 240 b +0.0724 × √ × a
1
2 n Pi ,
where ‘b’ and ‘a’ represented the mean section boundary length and mean sectional area, respectively. The
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Table 1 Coefficients of error (CE) for total volume, neuronal, and glial numbers as well as neuronal and glial densities in the mPFC
Groups Distilled water Olive oil Curcumin Sulfite Sulfite + curcumin
Neuron Density
Number
Density
Number
0.03 0.03 0.03 0.04 0.04
0.04 0.03 0.03 0.04 0.04
0.05 0.05 0.05 0.06 0.05
0.04 0.04 0.04 0.05 0.05
0.05 0.06 0.05 0.06 0.06
CE for the estimate of the total neuron number, CE(N), was calculated using CE(V) and CE(Nv) as follows: 1
CE(N) ) + CE (V ) 2, = CE (Nv − 2 n Q × CE(Nv) = 2 n−1 Q−
2
Glia
mPFC volume
2
1 2 2 Q− P (P )2 . + 2 − − Q P P The CEs have been shown in Table 1.
Estimation of the dendritic length Vertical uniform random sectioning is necessary for length estimation and it was achieved as follows.19–21,25,26 Briefly, 9–10 cylinders were punched out using a trocar with 1 mm diameter vertical to the pial surface of the cortex (Fig. 2). The cylinders were randomly rotated along their vertical axes and embedded in one paraffin block. Then, 100 μm thickness slabs were taken using a microtome. The mean dendritic length per neuron was calculated using the following formula: I N = Total dendritic length in the population . Total number of neurons in the population A fixed slab height of 100 μm was scanned using a microscope (Nikon E-200, Japan) equipped with an objective lens of 100× and numerical aperture of 1.4 connected to a computer. A cycloid grid and a counting frame were superimposed on the monitor image (Fig. 2). Overall, two quantities were measured: (i) number (Q −) of the cell bodies of the neurons using the optical disector method, and (ii) the total number of the intersections (I) between the dendrites axes and the cycloid oriented parallel to the vertical axis of the cylinder (Fig. 2). The following formula was used: I I N = 2 × a 1 M −1 , Q l asf −
a where ‘ ’ was the test area per cycloid test length, ‘asf’ l was the area associated with cycloid grid divided by the area of the counting frame, and ‘M’ was the final magnification at ×4000.26
Estimation of density and morphology of the dendritic spines To estimate the density and morphology of the spines, the above-mentioned dendrites were considered. Dendritic spines were identified as small protrusion that drawn out of the parent dendrite for less than 3 μm. Dendritic protrusion was classified as spines when they exhibited enlargement at the tip and presented stubby or mushroom-type forms. On the other hand, the spines without enlargement were defined as thin filopodia-like protrusions. The spines were counted only if they looked continuous with the parent dendrite. Density and morphology of the spines were quantified and stated as the number of spines per neuron.27
Statistical analysis The data were analyzed using non-parametric tests, including Kruskall–Wallis and Mann–Whitney U-test. Besides, P < 0.05 was considered as statistically significant.
Results Volume of the right mPFC A significant ∼8% reduction was observed in the total volume of mPFC in the sulfite-treated rats compared to the control ones (P < 0.005; Fig. 3). These findings demonstrated that concomitant treatment of curcumin during sulfite consumption prevented the mPFC volume loss induced by sulfite.
Neuron and glial cell counts (hemispheric) A significant difference was found among the study groups regarding the total number of neurons as well as the total number of glial cells (P < 0.005). Moreover, analysis of the mean neuron number revealed a significant decrease (∼15%) in the sulfite-treated group compared to the control groups (P < 0.005; Fig. 3). Likewise, the mean glial cell number was significantly lower (∼14%) in the sulfite group compared to the control groups (P < 0.005; Nutritional Neuroscience
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Figure 2 Estimation of the dendrites’ length. (A) The vertical cylinders were punched out from the MPF cortex perpendicular to its pial surface. (B) The cylinder was randomly rotated along its vertical axis, sectioned using a microtome, and mounted on a slide. (C) Four cycloids were placed at a rectangle. The length of each cycloid was identical to twice the length of the minor axis (r). The area associated with the cycloids was calculated by multiplying the X by Y. (D, E) When the sections were scanned, the number of the cell bodies of the neurons was counted using the optical disector method and unbiased counting frame. The total number of the intersections between the dendrites axes and the cycloid were counted (arrow head). The cycloid was oriented parallel to the vertical axis (arrow).
Fig. 3). The study results revealed that curcumin prevented neuron and glial cell loss induced by sodium metabisulfite.
Dendritic length measures On the average, a 10% decrease was observed in the length of dendrites per neuron in the rats treated with sulfite in comparison to those receiving distilled water, olive oil, or curcumin (P < 0.005). In addition, the concomitant treatment of sulfite + curcumin prevented the loss of dendritic length in rats (P < 0.005; Fig. 3).
Density and morphology of the spines The results showed that the total spine density was averagely reduced by ∼25% in the sulfite group in comparison to the control groups (P < 0.005; Fig. 4). Besides, the density of the mushroom spines was averagely reduced by ∼45% in the sulfite-treated rats in comparison to the control ones (P < 0.005; Fig. 4). Nonetheless, the stubby and thin spines of the mPFC were remained unchanged (Fig. 4). The concomitant treatment of sulfite + curcumin prevented the loss of dendritic spines in the mPFC.
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Discussion The present study evaluated the effects of treatment with sodium metabisulfite at the first step and showed a significant reduction in the volume of mPFC, a significant loss of neurons and glial cells, and reduction of dendritic length and spines in mPFC. Our earlier observations also showed that sodium metabisulfite could induce learning and memory changes in rats.8 Medial PFC has a key role in these tasks. Therefore, physiological changes are accompanied by histological alterations. Of course, neuron loss or physiological alterations are not limited to mPFC. One study reported that sulfite could cause the loss of hippocampal neurons in rats.3 Toxic effects of sulfite, both in vivo and in vitro, have been reported by various studies. For instance, a synergistic toxic effect on mesencephalic cell lines has been shown by sulfite and peroxynitrite anion.3 Moreover, sulfite treatment was revealed to cause impairment of the avoidance performance and increase the excitability of the spinal reflexes.6 Another study also reported that sodium metabisulfite caused neurotoxicity due to the increased lipid peroxidation in the brain tissue.3 A good justification for this neuronal
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Figure 3 The dot plot presents the total volume of the mPFC (A), total number of the neurons (B) and the glial cells (C), and dendritic length per neuron (D) in the mPFC in the rats receiving distilled water, olive oil, curcumin, and sulfite with or without curcumin treatment. Each dot represents the parameters in an animal.
toxicity of sulfite is that the brain tissue has low sulfite oxidase activity compared to other tissues. Another explanation is that extreme neurological abnormalities were observed in case of sulfite oxidase deficiency. Sulfite oxidase plays a protective role in detoxifying sulfite and low levels of expression of this enzyme may predispose neurons to damage by excess sulfite.5 There is little information about the mechanism of sulfite toxicity in the nervous tissue, but its toxic effect on the central nervous system may involve formation of sulfur and oxygen-centered free radicals.13 Previous reports suggested that various peroxidases could oxidize sulfite and also sulfite could be oxidized non-enzymatically. Non-enzymatic oxidation or oxidation by peroxidase of sulfite occurs via one electron oxidation. In this case, a sulfur trioxide radical is produced from sulfite during sulfite metabolism. It has been reported that sulfur trioxide radical may be involved in many toxic effects of sulfite, including destruction of amino acids, impairment of DNA synthesis, and increase of lipid peroxidation.28 In addition, it can be proposed that cysteine-S-sulfate, a brain damaging metabolite in sulfite oxidase deficiency, could be responsible for the observed toxic effects of sulfite. It should be mentioned that
cysteine-S-sulfate does not normally exist in human body.29 At the second step of the study, the protective effect of curcumin was evaluated. The findings showed that concomitant treatment of curcumin during sulfite consumption prevented the reduction in the volume of mPFC, loss of neurons and glial cells, and reduction of dendritic length and spines in mPFC. This finding is in line with our earlier observations showing that curcumin prevented learning and memory impairment induced by sulfite in rats.8 This finding is also in accordance with the previous studies reporting the neuroprotective effects of curcumin. Overall, there is a vast range of protective functions of curcumin in nervous system injuries, including improvement in lead-induced memory changes in rats, improvement in dorsal root ganglion and sciatic nerve structure and function after crush, improvement in behavioral changes after chronic stress in rats, and prevention of neuropathological changes in the hippocampus in an animal model of Alzheimer’s disease.14,30,31 Studies have also shown that different doses of curcumin decreased the oxidized proteins and interleukin1beta which is a proinflammatory cytokine increasing in the brains of these mice. Furthermore, treatment with curcumin was reported to improve the
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Figure 4 Density of the dendritic spines. The density of thin (A), mushroom (B), and stubby (C) spine per neuron and total spine density (D) in the mPFC of different groups are shown. The significant difference between the sulfite-treated group and other groups is indicated. *P < 0.005. DW: distilled water; OO: olive oil; CUR: curcumin; SUL: sulfite; SUL + CUR: sulfite + curcumin.
colchicines-induced cognitive impairment.14 Also, it could significantly reduce the colchicines-induced elevated lipid peroxidation. In summary, extensive studies have shown the protective effect of curcumin in almost all the bodily disorders due to its many useful properties, including anti-inflammatory, antihypertensive, anti-hyperlipidemic, anti-tumor, anticancer, anti-phlogistic, anti-diabetic, anti-psoriasis, anti-thrombotic, anti-hepatotoxic, and anti-microbial effects.9–11,14 Thus, the neuroprotective effects of curcumin against sulfite observed in our study might have resulted from its anti-oxidant, anti-inflammatory, anti-apoptotic, and anti-ischemic properties.
Conclusion Curcumin could prevent loss of neurons, glial cells, reduction of the total volume of the rats’ mPFC, and reduction of dendritic length and spines induced by sulfite ingestion. This finding agrees with the previous reports showing the neurotoxic effect of sulfite and neuroprotective effect of curcumin.
Acknowledgements This work was financially supported by grant no. 905954 from Shiraz University of Medical Sciences, Shiraz, Iran. The work was performed at
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Histomorphometry and Stereology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. This article was a part of the thesis written by Reza Asadi-Golshan, MSc student of Anatomy. Hereby, the authors would like to thank Ms A. Keivanshekouh at the Research Improvement Center of Shiraz University of Medical Sciences for improving the use of English in the manuscript. Source of financial support: This work was financially supported by grant No. 90-5954 from Shiraz University of Medical Sciences, Shiraz, Iran.
Disclaimer statements Contributors Designer and idea: Ali Noorafshan; Executive: Saied Karbalay-Doust; Animal experiment and stereological estimations: Reza Asadi-Golshan, Mohammad-Amin Abdollahifar. Funding None. Conflict-of-Interest None. Ethics Approval All the trial procedures conducted on the animals were performed in accordance with the standards established
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by the Animal Care and Ethics Committee at Medical School, Shiraz University of Medical Sciences, Shiraz, Iran (Agreement License No. 90-5954).
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