Original article
High fructose solution induces neuronal loss in the nucleus of the solitary tract of rats

Introduction

Simple sugars such as fructose with saturated fats are believed to be the major components of diet in many societies [27]. Carbohydrated soft drinks sweetened with fructose comprise about 33% of the total daily sugar intake in some drinks and foods. Fructose is used in several forms including high fructose corn syrup, sucrose, carbonated beverages, fruit products, cereals and other dairy products [20]. Unhealthy dietary habits can result in a heavy toll on the brain health. Overconsumption of fructose, particularly in the form of soft drinks, is increasingly [2] recognized as a public health concern [8]. A high fructose diet causes numerous pathological changes including oxidative stress, obesity, metabolic syndrome, glucose intolerance, insulin resistance, type II diabetes, liver disease, hypertension and cardiovascular disease [25].

It has been shown that consumption of fructose solution (15%) and a high fructose diet for six weeks can lead to an impaired cognitive function [2,22]. An intact plasma membrane is required for growth, repair and synaptic plasticity [2]. Researchers have claimed that fructose intake disrupts the plasma membrane of the neurons and the neuronal function might be affected [2]. It has been shown that consumption of a high caloric diet has harmful consequences for synaptic plasticity [31]. Also, a high caloric diet impairs the cognitive function, memory, dendritic spine density, synaptic plasticity [26,27] and neurogenesis in the hippocampus [30].

Although human and animal studies have proposed that excessive energy intake affects some regions in the brain adversely [27], the nucleus of tractus solitarius (NTS) has received less attention.

The NTS is a region in the brain that regulates and is involved in baroreflex sensitivity. Previous studies showed that the diet containing fructose could affect the morphology of the NTS. It was shown that a high fat and high carbohydrate diet taken for 30 days induced structural changes in the NTS and decreased the sensitivity of the baroreceptor reflex [3]. The structural changes were observed using transmission electron microscopy and including medullary sheath thickening, myelinated nerve atrophy and hyaloplasm dissolving. It has been shown that consumption of large quantities of fructose results in the impairment of baroreflex sensitivity and neurogenesis. Although the physiological and biochemical aspects of a high fructose diet on the brain have been previously studied, its effects on the quantitative aspects of the NTS have received less attention. The aim of the present study was to quantify the structural changes in the NTS following the consumption of high fructose solution in male rats using stereological methods.

Material and methods

Animals



Male Sprague-Dawley (150-180 g) 2.5-month-old rats were selected from the animal lab of the Shiraz University of Medical Sciences. All animal experiments were approved by the Animal Ethics Committee (approval license 90-5702) and kept in accordance with the university guidelines. Animals were weighed and distributed randomly into two groups (n = 6). They were kept at standard temperature (22-25°C) and in 12 h light-dark cycles. The rats had free access to tap water and fructose solution (10%) in the control and fructose-treated groups, respectively, for 6 weeks.



Intraperitoneal glucose tolerance test



On the last day of the experiment, the animals were kept fasting for 12, 0, 0 h and then anesthetized with ketamine and xylazine (70 and 5 mg/kg i.p., respectively). An unchallenged blood sample was checked at time of zero from the tail. Thereafter, 2 g/kg glucose was injected intraperitoneally and after 15, 30, 60, 90 and 120 minutes, blood samples were checked by the glucose meter (ACCU-CHEK Active, Germany) to determine the glucose concentration.



Biochemical analysis



After performing the glucose tolerance test, a sample of 2 ml of blood was obtained. Blood samples were centrifuged at 3000 γ for 10 min to obtain the plasma samples; then, glucose, triglyceride and cholesterol were quantified by the enzymatic method (Pars Azemoon Kit, IRAN) and plasma insulin level was assayed by rat insulin ELISA kit (Mercodia, Sweden).



Estimation of volume of the NTS



The brain was removed after transcardial perfusion-fixation with 2% paraformaldehyde in 0.1 M sodium phosphate buffer (pH: 7.2) for 20 min [23]. After histological processing, the brain stem was embedded in paraffin block.

The volume of the left NTS was estimated using Cavalieri method. Briefly, the rat medulla was sectioned exhaustively into 25 µm thick sections (t) using a microtome, from –11.04 to –15.96 mm from bregma [14,24]. About 8-12 sections in each animal were selected in a systematic random manner with a known fraction (k) (Fig. 1). The sections were stained with Cresyl violet. Using a microscope connected to a camera, the live image of each section was evaluated according to the rat brain atlas [24] at final magnification of 23× using stereological software designed at Histomorphometry & Stereology Research Centre (Shiraz University of Medical Sciences). The sum of the area of the sections (∑A) of the NTS was estimated using the software designed at the centre (Histomorphometry & Stereology Research Centre, Shiraz University of Medical Sciences). In addition, V(NTS) was estimated by the sum of points multiplied by the area per point using a point-counting method (Fig. 1). The area per point (a/p) was 0.02 mm² and 180 and 185 total points were counted on average per animal in the control and fructose-treated groups, respectively. Finally, the volume was estimated by:



V(NTS) = k × t × ∑A(NTS)



In the case of the second method, the formula was:



V(NTS) = k × t × ∑P(NTS) × (a/p)



Estimation of the total number of neurons



The numerical density NV (cell/NTS) or number of neurons in each unit volume of the NTS was estimated using the optical disector method, the position of the microscopic fields was selected by systematic uniform random sampling. An oil immersion on objective lens (100×) was used. An unbiased counting frame with inclusion and exclusion lines was superimposed on the images at final magnification of 3400× (Fig. 2). This frame avoids the “edge effect” and biased counting of the particles. The focal plane was moved downwards in z direction. A microcator was attached to the microscope to measure the z-axis travelling (depth). The guard zones were used to avoid cutting artifacts that occur on the upper and lower surfaces of the sections. Any counting event in focus within the upper (the first 5 µm) or lower guard zones was ignored. The distance between the guard zones was the “height of disector” which was 15 µm here. Any nucleolus 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. 2). The numerical density (NV) was estimated using the following formula:



NV(cell/NTS) = Q–/(p × a/f × h) × (t/BA)



here Q– was the number of the nucleoli coming into focus, p was the total counting of the unbiased counting frame in all fields, and h was the height of dissector. A point (P) lying at the centre of the counting frame was used to facilitate the counting of the frame area hitting the reference space. On the average, 160-400 cells were counted per NTS. Where a/f was the frame area, t was the real section thickness measured using the microcator, and BA was the block advance of the microtome which was set at 25 µm. Although in the present work we did not calibrate the microtome, it has been advised to calibrate the microtome to do a better evaluation [11]. The section thickness was measured at 6-10 fields of vision by sampling uniformly at random from each section and 72-120 location per animal. The mean section thickness was 20.3 µm in both groups. The total number of the neurons was estimated by multiplying the numerical density (Nv) by the V(NTS) [15,16,19].



Estimation of the coefficient of error (CE)



The CE for the estimate of the volume, that is CE(V), is the function of the noise effect and systematic random sampling variance for the sums of areas. When the cross-sectional areas “∑A” were estimated by the software, CE(V)was calculated using the following formula [17,18]:



CE(V) = (∑A)–1 × [1/12 × (3∑AiAi + ∑AiAi+2 – 4∑AiAi+1)]1/2



When the cross-sectional areas were estimated by point counting, CE(V) was calculated using the following formula [17,18]:



CE(V) = (∑P)–1 × [1/240 (3 × ∑PiPi + ∑PiPi+2 –

– 4∑PiPi+1) + 0.0724 × b/a1/2 × (n∑Pi)1/2]1/2



Where β and a represent the mean section boundary length and mean sectional area, respectively. The CE for the estimate of the total neuron number, that is CE(N), was derived from CE(V) and CE(Nv) as follows [6]:



CE(N) = [(CE2(Nv) + CE2(V)]1/2



CE(Nv) = [(n/n – 1) × [(∑(Q–)2/∑Q–∑Q–) +

+ (∑(P)2/∑P∑P) – (2∑ (Q– P)/∑ Q-∑P)]]1/2



Statistical analysis



Data were collected, analysed, and reported as mean, standard deviation (mean ±SD) and coefficient of variation (CV). Kruskall-Wallis and Mann-Whitney U-test and independent t-test were used to compare the differences between the groups. A p ≤ 0.05 was considered as statistically significant.

Results

Body and brain weights



The data showed no significant changes of these parameters in the fructose-treated rats in comparison to the controls (Table II).



Fluid intake



Analysis of data during 6 weeks showed that there was no difference between the two groups.



Biochemical analysis



The glucose, insulin, triglyceride and cholesterol did not show any significant differences between the two groups (Table I).



Glucose tolerance test



The data showed no significant difference between the two groups.



Volume of the NTS



The volume of the nucleus tractus solitarius was decreased by 41% on the average in fructose-treated rats when it was compared to the control groups (p ≤ 0.01) (Table II, Fig. 3).



Total number of the neurons



The results showed that the total number of the NTS neurons was significantly decreased 41% on the average in the fructose-treated rats in comparison with the control groups (p ≤ 0.01) (Table II, Fig. 4).



Coefficient of error



There is no difference between the prediction of coefficient of error (CE) for volume estimation of the NTS using the two described methods (areas estimation and point-counting methods).

Discussion

The present study evaluated the effects of consumption of high fructose solution for 6 weeks on the volume and number of neurons in the NTS of rats. The main goal of the present study was to assess the structural change of the NTS. The advantage of using stereological studies is obtaining unbiased and accurate estimations.

The weight of the animal and liquid intake were the first evaluated parameters. Some previous studies by Stranahan et al. and Brito et al. reported that high fructose diets induced weight gain and more drinking liquid in rodents [7,21,27]. Contrary to their claim, in this study, no weight gain and change in liquid intake was observed. The finding of the present research is in the same line with those of Takatori et al., Van der Borght et al. and Zamami et al. [28,30,32]. A probable description might be a different route of administration. Administration through drinking water may cause fewer amounts of fructose intake than food. Another reason might be a different species of experimental animals. Different species might show different levels of susceptibility after fructose treatment as a weight-gainer sugar. No difference was observed in the body weight, suggesting that obesity is not a major contributor to altered structural changes in the NTS.

Contrary to the findings of Takatori et al., Brito et al., Abdulla et al. and Catena, when consumed in drinking water in rats and hamster, fructose did not cause a significant impairment in glucose tolerance; this is in the same line with the results of the present study [1,7,9,28]. As listed earlier, different administration routes and different species of animals may explain the controversy observed [6,21,29].

In accordance with the present results, Van der Borght et al., Ueno et al. and Axelsen et al. [5,29,30] showed that consumption of fructose could not affect the insulin, free fatty acid and glucose levels in fasting state. The controversial findings are reported as well [4,7,27-29,32]. Alterations in circulatory parameters are accompanied by an increase in body weight since no difference was observed in the body weight; thus, no alteration in free fatty acid, triglyceride, fasting glucose and insulin is predictable.

The present study showed that the consumption of high fructose solution for 6 weeks reduced the volume of the NTS and number of the neurons without insulin resistance. The damaging effects of a high fructose diet extend directly to the brain [22], impairing the spatial memory in rats [25]. It was shown that male rats consuming fructose, in particular, have an increased amount of apoptotic cells in the dentate gyrus of the hippocampus and the impairment in neurogenesis [30]. However, by induction of insulin resistance, the cognitive function, structural plasticity and hippocampal function are impaired and dendritic spine density is reduced [27].

However, in the present study no change was seen in the insulin level. Therefore, other mechanisms can be responsible for the neuronal loss. Evidence is accumulating that neuronal cells can metabolize fructose. Thus, it is possible that fructose directly affects the brain as reported by Funari et al. [13]. The high fructose diets induce the toxic effects (e.g. hypophosphatemia, hyperuricemia) due to high fructose concentrations. Nocturnal hypertension and sympathetic nervous system changes due to a high fructose diet have been reported [12]. Impaired neurogenesis in the hippocampus by Van der Broght et al. is also reported [30]. Based on the results of these studies, fructose or one of its metabolites might induce loss of neurons in the NTS. Further research is suggested to assess the mechanism of fructose action on the neurons.

The modern stereological methods were applied to estimate the NTS volume and the numerical density of the neurons including Cavalieri and disector techniques. There are a limited number of research to show the volume of the NTS and their neuron number in rats. Dentremont et al. reported that the volume of the NTS was ~0.16 mm3 in the mouse [10]. They also reported that the total number of the neurons was ~87 000. They have used a modification of the Abercrombie method in contrast to our research which has relied on modern stereological methods.

Acknowledgement

This research was supported financially by the grant (No. 90-5702) from the vice chancellor of research of the Shiraz University of Medical Sciences. The stereological study was done in the Histomorphometry and Stereology Research Centre, Shiraz, Iran. This research is a part of the thesis of Enaiat Anvari, a PhD student of Physiology, Shiraz University of Medical Sciences.

References

 1. Abdulla MH, Sattar MA, Johns EJ. The relation between fructose-induced metabolic syndrome and altered renal haemodynamic and excretory function in the rat. Int J Nephrol 2011; 2011: 934659.

 2. Agrawal R, Pinilla FG. Metabolic syndrome’ in the brain: deficiency in omega-3 fatty acid exacerbates dysfunctions in insulin receptor signalling and cognition. J Physiol 2012; 590: 2485-2499.

 3. Ai J, Liang F, Zhou H, Zhao J, Wang N, Zhu S, Yang B. Mechanism of impaired baroreflex sensitivity in Wistar rats fed a high-fat and-carbohydrate diet. Br J Nutr 2010; 104: 291-297.

 4. Angelo GD, Elmarakby AA, Pollock DM, Stepp DW. Fructose feeding increases insulin resistance but not blood pressure in Sprague-Dawley rats. Hypertension 2005; 46: 806-811.

 5. Axelsen LN, Pedersen HD, Petersen JS, Holstein-Rathlou NH, Kjo/lbye AL. Metabolic and cardiac changes in high cholesterol-fructose-fed rats. J Pharmacol Toxicol Methods 2010; 61: 292-296.

 6. Braendgaard H, Evans SM, Howard CV, Gundersen HJ. The total number of neurons in the human neocortexunbiasedly estimated using optical disectors. J Microsc 1990; 157: 285-304.

 7. Brito JO, Ponciano K, Figueroa D, Bernardes N, Sanches IC, Irigoyen MC, De Angelis K. Parasympathetic dysfunction is associated with insulin resistance in fructose-fed female rats. Braz J Med Biol Res 2008; 41: 804-808.

 8. Brown CM, Dulloo AG, Yepuri G, Montani JP. Fructose ingestion acutely elevates blood pressure in healthy young humans. Am J Physiol Regul Integr Comp Physiol 2008; 294: R730-737.

 9. Catena C, Giacchetti G, Novello M, Colussi G, Cavarape A, Sechi LA. Cellular mechanisms of insulin resistance in rats with fructose-induced hypertension. Am J Hypertens 2003; 16: 973-978.

10. Dentremont KD, Ye P, D’Ercole AJ, O’Kusky JR. Increased insulin-like growth factor-I (IGF-I) expression during early postnatal development differentially increases neuron number and growth in medullary nuclei of the mouse. Brain Res Dev Brain Res 1999; 114: 135-141.

11. Dorph-Petersen KA, Nyengaard JR, Gundersen HJ. Tissue shrinkage and unbiased stereological estimation of particle number and size. J Microsc 2001; 204: 232-246.

12. Farah V, Elased KM, Chen Y, Key MP, Cunha TS, Irigoyen MC. Nocturnal hypertension in mice consuming a high fructose diet. Auton Neurosci 2006; 130: 41-50.

13. Funari VA, Crandall JE, Tolan DR. Fructose metabolism in the cerebellum. Cerebellum 2007; 6: 130-140.

14. Glatzer NR, Hasney PC, Bhaskaran DM, Smith BN. Synaptic and morphologic properties in vitro of premotor rat nucleus tractus solitarius neurons labeled transneuronally from the stomach. J Comp Neurol 2003; 464: 525-539.

15. Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Mo/ller A, Nielsen K, Nyengaard JR, Pakkenberg B, So/rensen FB, Vesterby A, West MJ. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 1988; 96: 857-881.

16. Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Mo/ller A, Nielsen K, Nyengaard JR, Pakkenberg B, So/rensen FB, Vesterby A, West MJ. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 1988; 96: 379-394.

17. Gundersen HJ, Jensen EB, Kieu K, Nielsen J. The efficiency of systematic sampling in stereology – reconsidered. J Microsc 1999; 193: 199-211.

18. Gundersen HJ, Jensen EB. The efficiency of systematic sampling in stereology and its prediction. J Microsc 1987; 147: 229-263.

19. Gundersen HJ. Stereology of arbitrary particles. A review of unbiased number and size estimators and the presentation of some new ones, in memory of William R. Thompson. J Microsc 1986; 143: 3-45.

20. Hanover LM, White JS. Manufacturing, composition, and applications of fructose. Am J Clin Nutr 1993; 58: 724S-732S.

21. Messier C, Whately K, Liang J, Du L, Puissant D. The effects of a high-fat, high-fructose, and combination diet on learning, weight, and glucose regulation in C57BL/6 mice. Behav Brain Res 2007; 178: 139-145.

22. Mielke JG, Taghibiglou C, Liu L, Zhang Y, Jia Z, Adeli K, Wang YT. A biochemical and functional characterization of diet-induced brain insulin resistance. J Neurochem 2005; 93: 1568-1578.

23. O’Kusky JR. Postnatal changes in the numerical density and total number of asymmetric and symmetric synapses in the hypoglossal nucleus of the rat. Brain Res Dev Brain Res 1998; 108: 179-191.

24. Paxinos G, Watson C. The Rat Brain: in Stereotaxic Coordinates. Elsevier, London 2007.

25. Ross AP, Bartness TJ, Mielke JG, Parent MB. A high fructose diet impairs spatial memory in male rats. Neurobiol Learn Mem 2009; 92: 410-416.

26. Stephan BC, Wells JC, Brayne C, Albanese E, Siervo M. Increased fructose intake as a risk factor for dementia. J Gerontol A Biol Sci Med Sci 2010; 65: 809-814.

27. Stranahan AM, Norman ED, Lee K, Cutler RG, Telljohann R, Egan JM, Mattson MP. Diet-induced insulin resistance impairs hippocampal synaptic plasticity and cognition in middle-aged rats. Hippocampus 2008; 18: 1085-1088.

28. Takatori S, Zamami Y, Yabumae N, Hanafusa N, Mio M, Egawa T, Kawasaki H. Pioglitazone opposes neurogenic vascular dysfunction associated with chronic hyperinsulinaemia. Br J Pharmacol 2008; 153: 1388-1398.

29. Ueno M, Bezerra RM, Silva MS, Tavares DQ, Carvalho CR, Saad MJ. A high-fructose diet induces changes in pp185 phosphorylation in muscle and liver of rats. Braz J Med Biol Res 2000; 33: 1421-1427.

30. Van der Borght K, Köhnke R, Göransson N, Deierborg T, Brundin P, Erlanson-Albertsson C, Lindqvist A. Reduced neurogenesis in the rat hippocampus following high fructose consumption. Regul Pept 2011; 167: 26-30.

31. Wu A, Ying Z, Pinilla FG. Oxidative stress modulates Sir2alpha in rat hippocampus and cerebral cortex. Eur J Neurosci 2006; 23: 2573-2580.

32. Zamami Y, Takatori S, Goda M, Koyama T, Iwatani Y, Jin X, Takai-Doi S, Kawasaki H. Royal jelly ameliorates insulin resistance in fructose-drinking rats. Biol Pharm Bull 2008; 31: 2103-2107.