Sagittal sections in the cerebral cortex show the pyramidal cells distribution (PYN) and neurocyte chromatolysis (arrow). (A, B) normal group. (C, D) diabetic group. (E, F) whey protein treated group. (Hematoxylin and eosin stain). 

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... for 30 min each. This was followed by two changes of xylene for 30 min each. The tissues were then impregnated with paraplast plus (three changes) at 60°C for 3 h and then embedded in paraplast plus. Sections (4 – 5 μ m) were prepared with a microtome, de-waxed, hydrated and stained in Mayer ’ s hemalum solution for 3 min. The sections were stained in eosin for 1 min, washed in tap water and dehydrated in ethanol as described earlier. Hematoxylin – eosin- stained sections were prepared [26]. For biochemical studies 0.25 g brain tissue was ho- mogenized in 3 ml of cold saline. The homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the clear supernatant was collected in a microfuge tube (0.5 ml each) and stored at -40C until used. Lipid peroxidation was determined by the reaction with thiobarbituric acid. Malondialdehyde (MDA) formed was determined according to the method of Preuss et al. [27]. Briefly, 1.0 ml brain supernatant was precipitated with 2 ml 7.5% trichloroacetic acid and centrifuged at 1,000 g for 10 min. Clear supernatant was mixed with 1 ml 0.70% thiobarbituric acid, incubated at 80 0C and the absorbance measured at 532 nm. Tetramethoxypropane was used as the standard. DPPH (2, 2-Diphenyl-1-Picryl Hydrazyl) is relatively stable free radical. The assay was carried out essentially by the method described by Joyeux et al. [28] and modified by Viturro et al. [29]. The bleaching rate of DPPH was monitored at 517 nm in the presence of the sample. In its radical form, DPPH absorbs at 517 nm, but upon reduction by an antioxidant or radical species its absorption de- creases. Briefly, tissue hydrolysate (1 ml) was added to a methanolic solution of DPPH (75 μ mol L − 1, 4 mL). The mixture was shaken vigorously and left in the dark at room temperature for 60 min, after which the absorbance was measured at 517 nm. The DPPH-scavenging effect (%) was calculated as [(absorbance at 517 control – absorbance at 517 sample)/OD517 control] × 100. α -tocopherol and BHT were used as controls. The Ugo Basile 47420-Activity Cage was used to record spontaneous co-ordinate activity in mice and variation of this activity in time. This test was performed 3 min for each animals. In this test, a mouse is placed on a horizontally oriented and mechanically rotating rod at 15 rpm. The rod is suspended above a cage floor, which is low enough not to injure the animal, but high enough to induce avoidance of fall. Mice naturally try to stay on the rotating rod, and avoid falling to the ground. The length of time that a given animal stays on this rotating rod is a measure of their balance, coordination, physical condition, and motor-activity [30]. The Ugo Basile 47200-Grip-Strength Meter suitable for mice automatically measures grip-strength (i.e. peak force and time resistance) of forelimbs in mice. The aim was to assess forelimbs muscle strength. Each animal tested three times and the peak force of each mouse was recorded. The mean of three values for each mouse was recorded. Blood glucose levels were determined using the Accu- Trend sensor (Roche Biochemicals, Mannheim, Germany). The Statistical Package for the Social Sciences (SPSS for windows version 11.0; SPSS Inc, Chicago) was used for the statistical analyses. Comparative analyses were conducted by using the general linear models procedure (SPSS, Inc). Also, the data were analyzed using one-way and two-way analysis of variance (ANOVA) followed by LSD computations to compare various groups with each other. Results were expressed as mean ± S.D. The level of significance was expressed as significant at P < 0.05 and highly significant at P < 0.01 [31]. The results showed that treatment of animals with WP significantly decreased glucose level in DM mice (Figure 1). The animals of WP group appeared more active and healthy during the behavioral test. In rotarod test, the WP treated mice stay on the rotating rod longer than the DM mice (Figure 2A). Treatment of mice with WP improved the balance, coordination, physical condition, and motor activity of the diabetic mice. In the activity cage, the DM group animals appeared anxious and recorded more scores in the horizontal and vertical activities than the normal and WP group of animals (Figure 2B). The WP group of animals showed significant improvements in the grip strength scores and recorded stronger beak than the DM group of animals (Figure 2C). DPPH content was significantly increased (P < 0.001) in DM group of animals. In WP-treated animals, the level of DPPH was reduced to the level of DPPH in normal animals (Figure 3A). WP treated mice showed insignifi- cant (P > 0.05) increase in lipid peroxidation whereas a significant increase in MDA (P < 0.001) was seen in DM group (Figure 3B). The normal cells of the cerebral cortex had spherical or pyramidal perikaryon whose nuclei were large with neurons arranged in a regular pattern (Figures 4A &B). The cerebral neurons appeared more developed toward the white matter (Figure 4). Pathological changes were observed in many sections in the DM group. Chromatolysis was observed in DM groups and WP treated animals showed significant neuronal protection. (Figures 4D & F). In the cerebellum, the numbers of neurons in the molecular layer of control mice (Figure 5A) were the higher compared to diabetic and WP group of animals. The control Purkinjee (PKC) cells were arranged in a single row of large neurons with pear-shaped perikaryon and large nucleus (Figure 5A). The lateral processes disappeared and the apical processes formed the permanent dendritic tree (Figure 5). In DM group (Figure 5B), some degenerated and pyknotic Purkinje cells were detected and some were more spindle- shaped and small (Figure 5C). The normal medulla neurons appeared large in size, varied in shape and had round nuclei (Figure 6A). In DM group, most of medulla neurons appeared small and pyknotic (Figure 6B). WP group medulla neurons showed improvement (Figure 6C). The results of this study demonstrate that hyperglycemia causes abnormalities in the neurobehavior of DM group animals such as physical balance, coordination and grip strength. Biochemical studies showed that DM is associated with disturbance in oxidative stress and neuronal pathology. Neuronal death may lead to cognitive deficits and an increased risk of brain complications [32]. In the diabetic animals, several brain alterations have been described, such as increased lipid peroxidation and DPPH radicals, neuronal changes in the cerebrum, cerebellum and medulla oblongata. Recently, a significant body of evidence to indicate that diabetes has detrimental effects on brain functions such as memory loss in type I and type II diabetes [33]. Some investigators have also reported a reduction in the length of the dendritic trees of the Purkinjee cells and pyramidal cells in diabetic ro- dents [34]. The diabetic animals show changes in dendritic morphology, probably associated with synaptic disturbances. This may explain memory and learning deficits [3]. Oxidative stress is widely accepted as playing a key mediatory role in the development and progression of diabetes and its complications, due to the increased production of free radicals and impaired antioxidant defenses [35]. Several mechanisms can contribute to increased oxidative stress in diabetic patients, especially chronic exposure to hyperglycemia. Accumulated evidence points out that hyperglycemia can lead to elevated ROS and reactive nitrogen species (RNS) production by the mitochondrial respiratory system [36], glucose autoxidation [37], activation of the polyol pathway [38], formation of advanced glycation end products [39], antioxidant enzyme inactivation and an imbalance of glutathione redox status [40]. Hyperglycemia can promote an important oxidative imbalance, favoring the production of free radicals and the reduction of antioxidant defenses. At high concentrations, ROS/RNS can damage the major components of the cellular structure, including nucleic acids, proteins, amino acids, and lipids [41]. Such oxidative modifications in the diabetes condition would affect several cell functions, metabolism, and gene expression, which in turn can cause other pathological conditions [42]. The oxidative stress leads to neuronal damage in several brain regions [5]. For example, neuronal loss in cerebrum impairs animal ’ s memory [43], neuronal loss in cerebellum can have effect on balance and coordination [6] and neuronal loss in medulla oblongata and spinal cord can affect physical activity of mice [44]. Supplementation with WP for 26 days decreased blood glucose and showed significant improvement in the physical balance, coordination, motor activities, and muscles strength in diabetic animals. WP supplement also decreased lipid peroxidation and DPPH radicals. Overall, this study demonstrated that WP supplementation significantly improved pathological alterations in diabetic mice as reported by Ebaid et al. [25]. WP has been found to significantly suppress hydroperoxide and ROS levels in liver and other tissues in mice by stimulating production of glutathione synthesis and thereby boosting cellular antioxidant defense [23]. Therefore, we suggest that WP may be an important therapeutic tool to combat oxidative stress-associated diseases [24]. We propose that WP may ameliorate diabetes in DM mice by its ability to neutralize free radicals and thereby prevent neuronal damage caused by oxidative stress. WP has a unique protective effect on glucose metabolism in STZ diabetic mice. WP supplementation improves the behavior of diabetic mice and reduces neuronal damage in the brain caused by oxidative ...